Aristotelia Chilensis Research Papers

Aristotelia chilensis: A Possible Nutraceutical or Functional Food

Jorge Peña Araos*

Faculty of Health Sciences, Pedro de Valdivia University, Santiago, Chile

*Corresponding Author:
Jorge Fernando Peña Araos
Clinical Managing Director
Universidad Pedro de Valdivia
Facultad de Ciencias de la Salud
Vicuña Mackenna Av. N° 40
Santiago, Región metropolitana 8320000, Chile
Tel: 56225197600
E-mail:[email protected]

Received date: July 21, 2015; Accepted date: August 19, 2015; Published date: August 24, 2015

Citation: Araos JP (2015) Aristotelia chilensis: A Possible Nutraceutical or Functional Food. Med chem 5:378-382. doi: 10.4172/2161-0444.1000289

Copyright: © 2015 Araos JP. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

Aristotelia chilensis (Maqui) is a native berry from Chile that has a wide range of biological actions. It is mainly composed of anthocyanins,indolealkaloidsand flavonoids. However, the high concentration of polyphenols has been identified as the main responsible for the anti inflammatory, analgesic and antioxidants effects of their extracts, juices and fruit. The nutraceuticals or functional foods development is an excellent opportunity to make a nutritious and healthy product. This review includes the chemical and nutritional characteristics of Maqui. The pharmacological and biological activities are included in this work. Also shows our need to develop new healthy products

Keywords

Aristoteliachilensis; Nutraceuticals; Antioxidants; Antidiarrheal; Anthocyanins

Introduction

A nutraceutical is a product made from a food, which besides having a nutritional role by providing nutrients and energy, is capable of delivering the body other active ingredients beneficial to health, differing from a functional food in the form of presentation and administration [1].

In recent years, both in the market of American nutraceuticalsas in Chile, there have been a number of products whose formulation containing Aristotelia chilensis. This fruit and leaves have shown a important range of biological actions, including antioxidants, antiinflammatory and analgesic effects. These effects have led to use these products as complementary therapeutic agents in the treatment of various chronic and degenerative disorders. The fruits of Aristotelia chilensis have a high content of anthocyanins, of indole alkaloids andflavonoids, which makes them an attractive resource to produce antioxidant extracts for use in functional foods or nutraceuticals [1,2].

The Aristotelia chilensis (Mol.) Stuntz, belongs to the family of Elaeocarpareae, a Chilean native species, popularly known as Maqui. It is a small tree that does not grow beyond 4-5 meters high, evergreen, with reddish stems, thin, flexible branches. Its bark is smooth and soft. Its leaves are stalked, lanceolate, with serrated edges, measuring 4-9 cm. It has small, whitish flowers arranged in axillary inflorescences. Its fruits are small berries of about 5 mm, bright blue or black color. The Maqui grows wild and is distributed from Limarí in Region IV to Region XI Aysén in Chile. It thrives in moist soil, invading ravines, slopes of hills and forest edges [1-4].

In Chile, this species is known for the use given to the fruit in the diet. However, little has been used for medicinal purposes [5]. Only native peoples (mainly Mapuches) have used its leaves as a tea for therapeutic purposes in the treatment ofdiarrhea, acutetonsillitis, throat and mouth ulcers [6].

In recent years there has been an active interest in the berries, which are a group of fruits red, violet or purple, with a high content of polyphenols. Among the highlights are cranberries, myrtle, raspberries, grapes, caulk and also maqui or blackberry [7].

Traditional Medical Purposes

Among the traditional medicinal uses, fruits and juices, as well as Maqui leaves have been used [1,6,8]. Internally, the fruits have been used as a tonic that has antidiarrheal, anti-inflammatory, healing, digestive, diaphoretic, expectorant, laxative and diuretic properties. Infusions of the leaves can be administered as gargling in inflammation of the oropharyngeal mucosa [8]. The Mapuches used the fruits of maqui externally to treat infected wounds and burn dry leaves as healing[1,6,8].

Nutritional Composition

In 1990 Schmidt-Hebbel et al. published tables of chemical composition of Chilean foods where maqui is included as a native fruit [9]:

Schmidt- Hebbel et al. Composición Química de Alimentos Chilenos, 8°Edición. Universidad de Chile. According to Damascos et al. the maqui contains a significant amount of ascorbic acid. Native shrub fruits had higher content of Br, Zn, Co, Cr, Fe, Mo, and Na than those of the exotic naturalized species The fruit nutrient content found in the studied species was similar or higher than other values reported for fruits of temperate and tropical species in the world [10].

Maqui mature leaves contain a lower amount of potassium than the young leaves and increased sodium content. The content of magnesium, chlorine, calcium, titanium and vanadium does not vary between leaves of different sites with different annual rainfall [1].

Chemical Composition

The Maqui has mainly anthocyanins, indole alkaloids, flavonoids, coumarins [11] caffeic and ferulic acid [12]. However, it has released the presence of gallic acid, gentisic, sinapic, hydroxybenzoic, vanillic, makonine, 8-oxo-9 dehydrohobartine and 8-oxo-9 dehydromakomakine [11,13].

Anthocyanins, flavonoids and phenolic acids are compounds which occur very often in many plant families. Their different biological activities, including antioxidant, antimicrobial, carcinogenic, cytotoxic, anti-inflammatory and mutagenic properties, make them interesting objects of research [2].

The most revealing anthocyanidins are 3-glycosides, 3,5-diglycoside, 3-sambubioside 5- and 3-glycosides sambubioside of cyanidin and delphinidin. The anthocyanidin at higher rates proved to be the delphinidin 3-glucoside sambubioside-5 (34% of total) [14,15].

The most important flavonoids are quercetin 5,3'-dimethyl ether, malvidin, petunidin, friedelin and ursolic acid. It has also been obtained in the methanol extract; quercetin 3-O-B-D- glucoside y kempferol [1,12,15].

Among the indole alkaloids identified include the following: aristone, aristoletine, aristotelinone, aristotelone, aristoteline, aristotelinine, protopine, serratoline, hobartinol, 8-oxo-9-dehydrohobartine, makonine, 8-oxo.9-dehydromakomakine, 9-dehydro-8- oxo-makomakine [16-18].

Pharmacological Activities

Antioxidant activity

Polyphenolic compounds maqui fruit have shown to possess potent antioxidant effects in vitro [19,20]. Maqui juices showed prevent lipid peroxidation induced by copper on human endothelial cells. The mechanism is explained by the effect of polyphenols maqui juice and by their antioxidant effect against oxidative stress [21].

Similarly, combining maqui juice with lemon juice important antioxidant effect against DPPH radicals, superoxide anion and hydroxyl were evidenced. The results suggested that lemon juice enriched with berries could be of potential interest in the design of new drinks with a nutritive related function on health for chronic diseases [22].

The new isotonic blends with lemon and anthocyanins-rich berries showed an attractive colour, especially in maqui samples, which is essential for consumer acceptance. Significantly higher antioxidant and biological effects were determined in the new blends, in comparison with the commercial isotonic beverages [23].

In general, aqueous and methanolic extracts of the fruit of the A. chilensis have shown antioxidant effects on in vitro models and xanthine oxidase inhibiting DPPH, fluctuating in the range of 52.9 and 62.7% respectively [12].

Crude extracts of leaves and fruits also showed antioxidant activity. Their polyphenols nhibit alpha-glucosidase enzyme. This enzyme is essential for the metabolism of carbohydrates and using these crude extracts, can be a useful mechanism in the management of diabetic patients. However, is necessary to make more research for warrants a potential dietary use of this berry in therapies to combat diabetes symptoms [24,25].

The Anthocyanins from A. chilensis improve fasting blood glucose levels and glucose tolerance in obese diabetic mice. Decreased glucose production and enhanced insulin-induced gene expression of glucose-6-phosphatase was observed in liver cells. For other hand, the Delphinidin 3-sambubioside-5-glucoside (D3S5G) was identified as principal anthocyanin of Maqui berry and responsible for these effects on glucose utilization [25,26].

The Aristoteline, aristone, serratoline and hobartinol were isolated from mixture of alkaloids (ALK-MIX). Ursolic acid, friedelin and quercetin 5,3'-dimethyl ether were present in the dichloromethane extract while quercetin 3-O-β-d-glucoside and kaempferol were present in the methanol extract. From aqueous extract (INFU) were isolated protopine, aristoteline and caffeic and ferulic acids. The effects of A. chilensis are here in demonstrated, validating its use intraditional medicine. Protopine was reported for the first time in Elaeocarpaceae for Muñoz in 2011[12].

Phenolics compounds are well-known antioxidants and their regular ingestion contributes to improve antioxidant status of plasma. Phytochemical findings describe phenolics compounds of leaves in Aristotelia chilensis (Molina) Stuntz, show that their administration in herbal teas may support the prevention of diseases whose etiology lies in the oxidative stress. The extracts obtained with polar solvents showed the stabilizing capacity of free radicals and the antioxidant capacity of plasma which, after taking A. chilensis tea, increased to 30.27% as an average. Herbal tea and extracts from A. chilensis exhibited antioxidant capacity under the described conditions [5]. The methanol extract (MeOH) from mature fruits of Aristotelia chilensis (Mol) Stuntz, showed antioxidant activities and cardioprotective effects on acuteischemia/ reperfusion in rat heart in vivo. On the other hand, the methanolic extract of the fruits was able to prevent theses harmful events in the animal’s heart by diminishing lipid oxidation. Antioxidant activities of MeOH were strongly correlated with total polyphenol content. Consistent with this finding, MeOH had the greatest ORAC and FRAP values as percentage of activity. These results show that these fruits could be useful as antioxidant, cardioprotective and nutraceuticals sources (Table 1) [27]. The presence of the compound indole-3-OH showed significant antioxidant effects and may explain the chemopreventive, anti-inflammatory and cardiopreventive activities this fruit [2].

CaloriesProteinCHOFibreAshCalciumPhosphorusIronPotasium
1500.8 g40.8 g0.8 g1.2 g87 mg44 mg30.5 mg296 mg

Table 1: Nutritional composition of Aristotelia chilensis in 100 g of edible part.

Anti-inflammatory and analgesic activities

Folk medicine attributes to the leaves of this species, analgesic and anti-inflammatory properties to be backed by scientific studies and has also determined that the leaves have antimicrobial and antioxidant activities [3,12,28]. The lack of effective and safe long-term therapies to treat the inflammatory process is an important problem in animal health. Thereby, the discovery of new anti-inflammatory alternatives is imperative. We have determined the anti-inflammatory effects of Aristotelia chilensis fruit concentrate in rats subjected to subplantar acute inflammation induced by carrageenan [29].

A bioguided study was carried out on serial extracts (hexane, dichloromethane, methanol, INFU and crude ALK-MIX). All extracts were evaluated for: a) topical administration against both arachidonic acid and 12-deoxyphorbol-13-decanoate (TPA)-induced inflammation in mice b) per-os administration against inflammation by deltacarrageenan- induced paw oedema in guinea-pigs and c) topicalanalgesiain tail flick andformalinmodel and per-os writhing test in mice. Greater anti-inflammatory effects were obtained against TPA with dichloromethane extract and methanol extract (63.9% and 66% respectively). INFU showed the most potent effect (56.2%) against arachidonic acid. Greater effects were obtained in the writhing test hexane and dichloromethane extracts (89.2%) [12].

In the topical analgesia models, all the extracts and ALK-MIX and the methanol extract were the most active (58.2 and 55.2%, respectively). In relation to the tail formalin assay, the methanol extract (74.1%) was the most active [12].

The proposed mechanism of action for analgesic activity of the hexane extracts, is considered which would be modulated by L-arginine serotonergic receptors (5-HT3), adrenergic (a-1), cholinergic-muscarinic (ACh-M) and via/NO (nitric oxide). Insteadantinociceptive activity of dichloromethane extract would be modulated by the opioid, serotonergic, adrenergic and nitridergic pathways. In the analgesic effect of methanol extract were involved the opioid systems, adrenergic, muscarinic cholinergic and L-arginine/NO. Alkaloids (Aristone, aristotelone, aristoteline and aristotelinine) demonstrated relax intestinal smooth muscle, explaining its anti-inflammatory and antispasmodic actions [3,28].

In general these compounds that occur in these Aristotelia species have been considered as the active principles of many anti-inflammatory plants. Thus, many phenolic acids, anthocyanins and flavonoids type have shown inhibitory activities on nitric oxide implicated in physiological and pathological process as chronic inflammation. These finding shows that the anthocyanins, flavonoids and phenolic acids may be responsible of the anti-inflammatory activity of this fruit. Actually, researchers are working in the kinetic of inhibition of these plant extracts and compounds as anti-inflammatory and they are dissecting the sites and mechanism of action as iNOS, Cyclooxygenase (COX), and TNF, among others [14].

Likewise, these extracts were able to reduce the production of nitric oxide (3.7-25.5%) and prostaglandin E2 (9.1-89.1%) and the expression of inducible oxide nitric synthase (9.8-61.8%) and cyclooxygenase-2 (16.6-62.0%) in RAW 264.7 macrophages stimulated by lipopolysaccharide [30].

Anthocyanins have effect during the inflammatory process, since they act by inhibiting the COX. Besides its antioxidant purpose and it inhibits adhesion of leukocytes to endothelial reaction to suppress the expression of molecules VCAM cells. They also inhibit mast cell degranulation and decreased levels of IL-2, IFN-y, and TNF-α [31].

It has been shown that anthocyanins inhibit the secretion of inflammatory cytokines such as IL-8, MCP-1, IL-1B-1 in neutrophil chemotactic cytokine-induced and IL-6 in cell and animal model after an inflammatory stimulus [32,33]. This suggests that it is feasible to use a formulation of dermatological use with A. chilensis to treat inflammatory skin diseases such as rosacea, sunburn and more circumscribed inflammatory lesions [34]. Other studies have suggested that supplementation with anthocyanins in vivo inhibits activation of NF-kB regulates many pro-inflammatory chemokines, cytokines and inflammatory mediators [35].

Anthocyanins exhibit a wide range of antioxidant protection and beneficial therapeutic effects including genomic DNA integrity, cardiovascular protection, neurological protection, anti-inflammatory and anti-cancer and can significantly reduce the production of NO, reducing oxidative stress in the anti-inflammatory process [29].

Others biological activities of general interest

A few years ago, the effects of concentrated of A. chilensis on the expression of COX-2, their signaling pathways and cell viability of colon cancer was assessed. Treatment of Caco-2 cells with A. chilensis for 24 hours reduced the expression of the protein and mRNA of COX- 2, and reduced the luciferase activity regulated by NF-κB or NFAT.

Treatment of Caco-2 cells for 4 hours with A. chilensis temporarily reduced IκBα cytoplasmic levels, increased phosphorylation of ERK1/2 and Akt and the expression of c-fos. Also cell viability at concentrations that reduced expression of COX-2 was not affected. These results suggest a potentialanticancer and anti-inflammatory effect [36].

On the other hand, the tannins of the fruit of the maqui have astringent properties and anti-diarrheal and the polyphenols of the leaves showed effects anti-hemolytic in vitro [24].

An experimental study evaluated the neuroprotective potential of an extract made from maqui polyphenols in Alzheimer's disease model induced by soluble oligomers B-amyloid.

Neurons cultures of rat hippocampus were used and neuroprotection was observed when neurons were incubated with amyloid B (0.5 uM) more maqui extract for 24 hrs. In the mechanism of action was observed a recovery in the frequency transients calcium channel, compared with the control group receiving only B-amyloid (Only B-amiloide=72 ± 3% and B-amiloide+Maqui= 86 ± 2%; n=5). This was positively correlated with observed in spontaneous synaptic activity and the preservation of dendritic branches changes. The antioxidant activity of maqui is primarily responsible for these changes [37]. Alzheimer's disease is characterized by the presence of different types of extracellular and neurotoxic aggregates of B-amyloid. Recently, bioactive compounds extracted from natural sources showingneuroprotective properties have become of interest in brain neuroregeneration. The results suggest that the maqui induces changes in the aggregation kinetics of Aβ producing variations in the nucleation phase. In conclusion, the maqui induces a potent neuroprotection by direct interaction with the B-amyloid aggregates, generating far less toxic species and in this way protecting the neuronal network [37].

The use of methanol extract of the ripe fruit provides cardioprotective activity in models of acute ischemia reperfusion in an in vivo study in rats [27]. Metabolically, has been evaluated in vitro the ability the some extracts of maqui, in reducing adipogenesis and lipid accumulation in adipocytes. In this regard, the phenolic extracts showed inhibit lipid accumulation between 4 to 10.8% in mature adipocytes and between 5.9 and 37.9% in those in differentiation state [1,38]. Concentrated juice of Aristotelia chilensis has a high content of phenolic compounds with antioxidant capacity, which protect both LDL from oxidation and endothelial cells from intracellular oxidative stress, suggesting that Aristotelia chilensis could have anti atherogenic properties [36].

Obesity is characterized by an increase in the infiltration of monocytes into the adipose tissue, causing an inflammatory condition associate with, for example, the development of insulin resistance. Thus, anti-inflammatory based treatments could emerge as a novel and interesting approach. It has been reported that Chilean native fruits maqui (Aristotelia chilensis) and calafate (Berberis microphylla) present high contents of polyphenols, which are known for their antioxidant and anti-inflammatory properties. The extracts of theses fruits present important inhibitory-like features over the inflammatory response of the interaction between adipocytes and macrophages, comprising a potential therapeutic tool against comorbidities associated with obesity development [39].

One study showed the protective effect of topical formulations containing quercetin on damage caused by oxidative stress induced by ultraviolet radiation. Two different formulations were used; a nonionic emulsion with high lipid content and other anionic emulsion with low concentration oflipids. The results showed that the inflammatory damage induced by ultraviolet radiation decreased with both formulations, with better results when using non-ionic formulation rich in lipids [34].

 

In search of a nutraceutical formulations or a functional food based on A. chilensis

Infusions of its leaves have long been used in the traditional native herbal medicine to treat different ailments. Phytochemical studies of chemical composition of the plant indicate the presence of indolic alkaloids, flavonoids, cianidine glucosides, delfidine, malvidine, petunidine, cumarines and triterpenes. These compounds, particularly the flavonoids, have antioxidant properties. In order to evaluate the mechanisms of its toxicity and their antioxidant properties, the leaves aqueous extracts were induced to interact with human red cells, their isolated unsealed membranes (IUM), and molecular models of the human erythrocyte membrane. Results of the study indicate that aqueous extracts of A. chilensis induced an alteration ofhumanerythrocytemorphology from the normal discoid shape to an echinocytic form, changes that are explained in terms of the extract interaction with the membranes outer phospholipid monolayer .

In recent years, the food industry has shown great interest in developing maqui juice for its extraordinary potential as an antioxidant and functional food. In this sense, the development of a nutraceutical made from A. chilensis seems a logical step to have a product rich in polyphenols, flavonoids and anthocyanins [40].

For the evaluation of products and chemical characterization of the nutrients and healthy compounds, the quality of the product in dry and fresh should consider [41,42].

1) Drying and juice production substantially affected the stability of maqui anthocyanins

2) Stability of cyanidin and delphinidin derivatives differed during processing.

3) Maqui pomace was a valuable source for obtaining polyunsaturated fruit oils.

4) Maqui products exhibited nutritionally relevant mineral and trace element levels.

Increased domestic and international demand of maqui since 2006, increasing from 733 kg to more than 50,000 kg in 2011 (data from Chile Foundation 2012) showing the interest in this fruit [7].

The maqui has been used to make jams, fresh juices with water and sugar or functional foods by providing a number of compounds potentially beneficial for the health and rich in Polyphenolics components. The physical and chemical characteristics of the maqui juice with and without added sugar are showing in the Table 2 (from a study by Araneda et al. at the School of Agronomy of the Catholic University of Temuco, Chile) [40].

ParameterJuice with sugarJuice without sugar
Soluble solids (°Brix)22 ± 1.8714 ± 1.79
pH3.78 ±1.033.35 ± 0.23
Acidity (%)0.95 ± 0.130.85 ± 0.14
Humidity (%)75.22 ± 1.4986.6 ± 0.84
Drymatter (%)24.78 ± 0.2713.4 ± 0.13
Ash (g)0.51 ± 0.020.44 ± 0.04
Sacarose (g 100mL-1)19.32 ± 1.7116.57 ± 0.65
Reductedsugars (g 100mL-1)39.66 ± 1.906.8 ± 0.22
Total invertsugar (g 100mL-1)58.98 ± 1.9823.37 ± 0.69
Crudeprotein (%)0.40 ± 0.010.19 ± 0.01
Total Polyphenols (mg 100 mL-1 EAG)829.22±25.86993.21±54.87
Total Polyphenols (mg EAG g-1 MS-1)33.46 ± 2.2274.1 ± 1.08
Total Carbohydrates (%)3.41±0.471.51±0.44

Table 2: Physical-chemical analysis obtained maqui juice with and without sugar (40).

Evidently, the total polyphenols content is not always the same, can be higher or lower depending on the ripeness of the fruit at harvest time, the type of climate, storage conditions and genotype[4,43].

The maqui is characterized as an important source of phenolics compounds [24] and a product with a high content of polyphenols of natural origin, is a healthy product and considered functional [44].

The fruit of the maqui is considered the healthiest berry exotic, because of its particularly high concentration of bioactive polyphenols [27,38] and the use of this fruit rich in polyphenols in preparing of other foods, showed a reduction in the use of artificial additives antioxidants obtaining thus healthier foods [44,45].

The polyphenols, can prevent heart disease, by possessing vasodilatory effects, antibiotics, anti-inflammatory, anti-apoptotic, anti-atherogenic. Also could be prevent colon cancer [36,46], and providing a possible protective effect in preventing diseases of the retina [47]. Regardless of the course to follow; develop functional foods or nutraceuticals, the Maqui is a super food capable of providing innovative alternatives in food and natural product for health.

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  37. Fuentealba J, Dibarrart A, Saez-Orellana F, Fuentes-Fuentes MC, Oyanedel CN, et al. (2012) Synaptic silencing and plasma membrane dyshomeostasis induced by amyloid-B peptide are prevented by Aristoteliachilensis enriched extract.J Alzheimers Dis 31: 879-889.
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Abstract

Maqui (Aristotelia chilensis [Molina] Stunz) is a small dioecious tree native to South America with edible fruit characterized by very high antioxidant capacity and anthocyanin content. To preserve maqui as a genetic resource it is essential to study its genetic diversity. However, the complete genome is unknown and only a few gene sequences are available in databases. Simple sequence repeats (SSR) markers, which are neutral, co-dominant, reproducible and highly variable, are desirable to support genetic studies in maqui populations. By means of identification and characterization of microsatellite loci from a maqui genotype, using 454 sequencing technology, we develop a set of SSR for this species. Obtaining a total of 165,043 shotgun genome sequences, with an average read length of 387 bases, we covered 64 Mb of the maqui genome. Reads were assembled into 4,832 contigs, while 98,546 reads remained as singletons, generating a total of 103,378 consensus genomic sequences. A total of 24,494 SSR maqui markers were identified. Of them, 15,950 SSR maqui markers were classified as perfects. The most common SSR motifs were dinucleotide (31%), followed by tetranucleotide (26%) and trinucleotide motifs (24%). The motif AG/CT (28.4%) was the most abundant, while the motif AC (89 bp) was the largest. Eleven polymorphic SSRs were selected and used to analyze a population of 40 maqui genotypes. Polymorphism information content (PIC) ranged from 0.117 to 0.82, with an average of 0.58. Non-significant groups were observed in the maqui population, showing a panmictic genetic structure. In addition, we also predicted 11150 putative genes and 3 microRNAs (miRNAs) in maqui sequences. This results, including partial sequences of genes, some miRNAs and SSR markers from high throughput next generation sequencing (NGS) of maqui genomic DNA, constitute the first platform to undertake genetic and molecular studies of this important species.

Citation: Bastías A, Correa F, Rojas P, Almada R, Muñoz C, Sagredo B (2016) Identification and Characterization of Microsatellite Loci in Maqui (Aristotelia chilensis [Molina] Stunz) Using Next-Generation Sequencing (NGS). PLoS ONE 11(7): e0159825. https://doi.org/10.1371/journal.pone.0159825

Editor: Yuepeng Han, Wuhan Botanical Garden of Chinese Academy of Sciences, CHINA

Received: April 16, 2016; Accepted: July 9, 2016; Published: July 26, 2016

Copyright: © 2016 Bastías et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: Raw data are available from NCBI-SRA (SRA accession: SRP073490).

Funding: This work was supported by grants from Alto Cachapoal Scientific Research Fund (Pacific Hydro Chacayes, Chile), and Fondecyt 1121117. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Maqui (Aristotelia chilensis [Molina] Stunz) is a small dioecious tree native to South America that belongs to the Elaeocarpaceae family. It ranges from 3 to 4 m in height and grows in altitudes of up to 2,500 m.a.s.l. Maqui grows best in rich moist topsoil on hillsides or at forest edges. It is a pioneer species that colonizes newly grazed land, forming monospecific associations [1]. Maqui is an evergreen plant with serrated lanceolate leaves with a leathery texture and a reddish petiole. The fruit is a shiny black berry, 3–5 mm in diameter, which is used as food, dye and medicinal remedy [1–3].

Maqui fruit has analgesic, anti-inflammatory and antioxidant properties [4–6] and is characterized by high levels of antioxidant activity due to elevated anthocyanin and polyphenol content [4,7–9]. Maqui extracts have applications as food additives and nutraceutics because of the plant’s anthocyanin and polyglycosides content [10,11]. The fruit is collected from wild plants and overharvesting is threatening wild populations of A. chilensis. To preserve maqui as a genetic resource it is essential to study the genetics of this species. A better understanding of the genetic relationships among individuals and populations is needed for effective planning to preserve wild populations, as well as to estimate the genetic potential of traits with economic interest to breeding programs.

The genetic diversity of a population allows it to respond and adapt to environmental changes [12]. At the molecular level, genetic diversity in the form of variations in nucleotides, genes and genomes can be evaluated among different accessions of the same population (intraspecific) or among populations (interspecific) [13]. Genetic variation of a species is the fundamental basis of the evolution, adaptation and development of crop varieties [14]. Genetic variation emerges from the differences in DNA sequences (insertion, deletion, duplication or inversion). These differences can be functional, causing changes in metabolic or phenotypic characters, or neutral [15].

DNA molecular markers are powerful tools for analyzing genetic diversity. They are based on DNA sequence polymorphisms [16]. DNA molecular markers show Mendelian inheritance, ensure wide coverage of the genome and are neutral to environmental factors and stages of development as they are not affected by other genes or factors [15,17]. According to the development of technologies in molecular biology, DNA markers can be divided into three broad categories: first, second and third generation markers.

First generation markers are based on DNA-DNA hybridization, such as restriction fragment length polymorphism (RFLP). Second generation markers are based on DNA fragment amplification by PCR such as cleaved amplified polymorphic sequences (CAPs), amplified fragment length polymorphism (AFLP), sequence-characterized amplified regions (SCARs) and simple sequence repeats (SSRs), among others. Finally, third generation markers like single nucleotide polymorphisms (SNP) depend on a meaningful analysis of next-generation sequencing (NGS) data. Moreover, there are genome-scanning platforms based on array technology, such as expressed sequence tags (EST) and diversity arrays technology (DArT) that facilitate genome analysis [13,16,17].

The use of first generation markers has been drastically mainly because of limits on the number of samples that can be handled. At the same time, the high costs of third generation markers like SNPs and their derivative platform of arrays restricts their use in large-scale genotyping. In contrast, second generation PCR-based markers like SSRs are considered highly cost/effectivity [13,16].

Microsatellites, namely simple sequence repeats (SSRs), short tandem repetitions (STRs) and short sequences length polymorphisms (SSLPs), are short (1–6 bp) tandemly repeated DNA sequences [18]. SSRs are neutral, co-dominant, reproducible and highly variable molecular markers [19]. SSRs are found mainly in noncoding DNA regions whose origins and functions are unclear [20,21]. Microsatellites may generate genetic variation in the genome and influence transcriptional activity in promoter regions [22].

Based on the type of repeat sequence, microsatellites can be classified into three categories, perfect, imperfect and compound/composite, as defined by Weber (1990) [23]. However, other authors have defined distinct categories [21,24]. Perfect microsatellites are uninterrupted series of a repeat unit, e.g. (AT)15, while imperfect microsatellite sequences differ from perfect ones by the presence of one to three base interruptions in the run of tandem repeats, e.g. (AT)10C(AT)8. Finally, the sequence of a compound or composite microsatellite contains two adjacent distinctive sequence-repeats, e.g. (AT)7(TG)10. In general, the degree of polymorphism increases with the total length of the SSR. Longer and perfect SSR loci are known to exhibit greater allelic variability [25,26]. Significantly higher levels of genetic variation are found in perfect SSRs than in imperfect SSRs [27]. SSR markers have been used in many research areas such as linkage mapping [28], genetic diversity [29], phylogenetic analysis [30], genotype identification [31] and comparative genomic research [32].

SSRs have to be isolated and characterized for each species. Three general methods have been described for SSR isolation [13]: (i) the standard method, where a library is developed of genomic DNA, cDNA and PCR fragments [33], (ii) the automated method, where SSR sequences are searched in sequence databases and (iii) next generation sequencing (NGS), where the total or partial genome is sequenced using massive sequencing [34]. New massive sequencing platforms have enabled SSR sequencing from DNA or RNA sequencing. With these technologies it is not necessary to create a library as large numbers of sequences are rapidly produced. At the same time, the costs involved are decreasing [35–39].

In our work, we used high throughput next generation sequencing (NGS) to identify microsatellite or simple sequence repeat (SSR) markers from the maqui (A. chilensis) genome. In addition, partial sequences of genes and some regulator factors of maqui are described. This will constitute the first public platform to facilitate genetic and molecular studies of this promising species.

Materials and Methods

Plant Material

Young maqui (A. chilensis) leaves were collected at the Río Los Cipreses National Reserve (O’Higgins Region, Chile) and other locations in Chile (Fig 1, S1 Table). Samples were frozen in liquid nitrogen and stored at −80°C until DNA extraction and subsequent analysis.

Fig 1. Sampling of maqui (A. chilensis) in Chile.

Samples of maqui were collected from Santiago (33°25'0"S 70°39'0"W) to Los Lagos (40°34'57.41”S 73°43'55.80”W) of Chile. A is an aerial map of Chile. B is an enlargement of the sampled area, and C corresponds to sampling at the Río Los Cipreses National Reserve (O’Higgins Region, Chile). The original pictures were taken from USGS The National Map, http://viewer.nationalmap.gov/ (Coordinates of sampling are described in S1 Table).

https://doi.org/10.1371/journal.pone.0159825.g001

DNA sequencing

Genomic DNA (approximately 100 mg) was extracted from leaves of one maqui plant (A. chilensis) named T1S (Fig 1, S1 Table) with the DNeasy Plant Mini Kit (Qiagen Inc., Valencia, CA, USA) following the manufacturer’s protocols. DNA quality and quantity were checked by agarose gel electrophoresis and spectrophotometric measurement of UV absorption at wavelengths of 260 and 280 nm and absorbance ratios of 260/280 and 260/230 using an Infinitive M200Pro Nanoquant (Tecan Group US, Inc., Morrisville, NC, USA).

The DNA was subjected to shotgun pyrosequencing (1/8th run) on the Roche 454 Genome Sequencer FLX at Macrogen Inc.(Seoul, Korea) using GS-FLX Titanium reagents (Roche Applied Science), as has been described by Margulies et al. (2005) [40]. GS FLX data processing was performed using the Roche GS FLX software (v 2.9). The obtained nucleotide sequence reads were assembled with GS De Novo Assembler (v 2.9) software.

SSR detection and primer design

We only analyzed perfect SSRs. The contig and singleton sequences obtained in FASTA files were screened for di-, tri-, tetra-, penta-, hexa-, hepta- and octanucleotide repeats, and only dinucleotide repeats with lengths of ≥ 6, trinucleotide repeats with lengths of ≥ 4, and tetranucleotide to octanucleotide repeats with lengths of ≥ 3 were accepted using MIcroSAtellite identification software [41]. The program allows for direct primer design using PRIMER 3 [42] by searching for microsatellite repeats and primer annealing sites in the flanking regions.

SSR Validation

Primers were successfully designed and synthetized for a total of 50 SSRs (at least three repeats) from sequencing data. A subset of DNA of ten individual maqui plants was prescreened for the quality of amplified SSRs by capillary electrophoresis on a fragment analyzer (Advanced Analytical Technologies, USA). We selected the polymorphic SSR loci with scorable polymorphic bands. The remaining loci that showed no amplification, multibanding patterns, or monomorphic bands were discarded. Finally, to characterize the confirmed loci, a total of 40 maqui (A. chilensis) individuals were sampled from the Rio Los Cipreses National Reserve and other locations in Chile (Fig 1, S1 Table).

The genomic DNA was extracted from the leaves of each sample using the DNeasy Plant Mini Kit (Qiagen Inc., Valencia, CA, USA) following the manufacturer’s instructions. The polymerase chain reaction (PCR) was carried out in a final volume of 25 μl containing 20 ng of genomic DNA, 2.5 μl 10 x Master mix (Stratagene Paq5000 DNA Polymerase buffer; Agilent Technologies, Santa Clara, CA, USA), 0.4 μl 10 mM dNTPs, 0.4 μl 10 uM of each forward and reverse primer, 1 U of Taq polymerase (Stratagene Paq5000 DNA Polymerase; Agilent Technologies, Santa Clara, CA, USA) and distilled nuclease-free water up to a final volume of 25 μl/tube with the following temperature profile: an initial 5 min at 95°C, 35 cycles of 45 s at 95°C, 80 s at an annealing temperature in accordance with the primers (S2 Table), 45 s at 72°C, and finally, 5 min at 72°C. PCRs were performed using a Swift Maxi Thermal Cycler model MX-BLC7 (Esco, Hatboro, PA, USA). The amplification products were separated by capillary electrophoresis using a Fragment Analyzer™ 12-capillary Automated CE System (Advanced analytical Technologies, Ames, IA, USA) using DNF-900 double-stranded DNA Reagent Kit (Advanced Analytical Technologies, Ames, IA, USA). The gel, inlet buffer, capillary conditioning solution, 35bp/500 bp marker and instrument were prepared according to the manufacturer’s instructions. To prepare the sample plate, 4 μl of each DNA sample and 22 μl of 1X TE dilution buffer were placed in the respective wells of the sample plate and first vortexed at 3000 rpm for 2 min and then centrifuged. The sample plate was run immediately after being prepared. To run the samples, the plate was placed in one sample plate tray of the Fragment Analyzer™. The DNF-900 dsDNA Reagent Kit experimental method (12-Capillary, 50–80 array) was loaded and run.

Data analysis of putative alleles

After capillary electrophoresis separation, the data was opened and processed using the software PROSize® 2.0 version 1.3.1.1 (Advanced Analytical Technologies, Inc., Ames, IA, USA). The data were normalized to the lowest (35 bp) and highest markers (500 bp), and calibrated to the 75–400 bp Range DNA ladder. A peak was considered a putative allele when the percentage of the area under the curve was more than 10%, with a maximum of two counted alleles per individual.

A double-entry matrix was developed with all counted alleles. Simple sequence-repeat alleles were scored as present (1) or absent (0). An index of similarity among pairs was developed, as well as a neighbor-joining method to generate a dendrogram. A bootstrap of 1000 was included to evaluate the robustness of the distribution tree. All analyses were performed with DARwin 6.0.012 software [43].

Polymorphism information content (PIC)

The PIC values of all the polymorphic SSR markers were calculated as follows: , where k is the total number of alleles detected for a given marker locus and Pi is the frequency of the ith allele in the set of investigated genotypes [44].

Putative maqui gene prediction

Putative genes were predicted with AUGUSTUS software [45], analyzing contig and singleton genomic sequences from maqui. The program is based on a hidden Markov model and is used for the ab initio prediction of protein coding genes in eukaryotic genomes. Arabidopsis thaliana (L.), Heynh. was used as the model organism. BLAST2GO was then used to functionally annotate potential coding sequences or predicted genes [46]. This research tool was designed to allow consistent gene annotation and Gene Ontology (GO) based data mining of sequence data for which GO annotation is not yet available. The sequences were analyzed with Blastx tool against a base of customized local database with sequences of different plant species, giving a description of the best hit of mapped sequence, GO terms, enzyme commission (EC) number and InterPro partners. The sequences were identified in a non-redundant database with a personalized e-value cutoff of E -6.

Predicting microRNAs

Potential microRNAs were computationally predicted using a hidden hierarchical Markov model (HHMMIR) [47], which allows de novo prediction of microRNAs using hidden Markov hierarchical models. These results were validated using miPred [48] and miRdup [49] tools, with which respectively we distinguished the real pre-miRNAs from other hairpin sequences with similar stem-loops and predicted the location of microRNAs in their pre-miRNA. Plant miRNAs described in the miRbase database [50] were used as models.

Results

SSR detection from NGS sequence analysis

SSR markers can be developed using next generation sequencing for species like maqui (Aristotelia chilensis), which lack genomic data [35]. In our study, the genomic DNA of maqui was partially sequenced by the de novo shotgun 454 pyrosequencing. A total of 165043 bp pair-end reads were obtained with an average read length of 408 bp, covering 64 Mb of the maqui genome (Table 1).

Reads were assembled into 4832 contigs, while 98546 reads remained as singletons, generating a total of 103378 consensus genomic sequences (Tables 1 and 2). About 80% of contigs were assembled with fewer than 30 reads. The average GC content of genomic maqui DNA was 38.94%.

Identifying maqui SSR in genomic sequences.

With the goal of determining a set of genetic markers in maqui (A. chilensis), contigs and singleton sequences were surveyed for the presence of SSRs by means of the MIcroSAtellite (MISA) tool [41]. The total of identified SSR sequences was 24494 (Table 2, S3 Table). S3 Table shows the putative SSR markers and their possible primer pairs. However, only perfect SSRs were considered in the following analysis, with a repeat motif size range of 2 to 8 bp and a length of >12 bp. This includes dinucleotide repeats ≥ 6, trinucleotide repeats ≥4, and tetra-, penta-, hexa-, hepta- and octanucleotide repeats ≥3. All the mono-nucleotide repeat SSRs were excluded from this analysis. A total of 15950 SSRs were identified, which were contained in 13531 contigs and singleton sequences (Tables 2 and 3).

Distribution of SSR motif lengths and types and repeat numbers.

We examined the distribution of maqui microsatellites with regard to motif length and type and the number of repeats (Figs 2 and 3, S4 and S5 Tables).

Fig 2. Frequency distribution of SSR loci of maqui (A. chilensis) by motif length.

The graph is based on a total of 15950 SSR markers detected in non-redundant genomic maqui DNA. Di, tri, tetra, penta, hexa, hepta and octa refer to dinucleotides, trinucleotides, tetranucleotides, pentanucleotides, hexanucleotides, heptanucleotides and octanucleotides, respectively.

https://doi.org/10.1371/journal.pone.0159825.g002

Fig 3. Distribution of SSR from maqui (A. chilensis) with Di- to Octa-nucleotides by repeat numbers.

The graph is based on a total of 15950 SSRs detected in non-redundant genomic maqui DNA. Di, tri, tetra, penta, hexa, hepta and octa refer to dinucleotides, trinucleotides, tetranucleotides, pentanucletides, hexanucleotides, heptanucleotides and octanucleotides, respectively.

https://doi.org/10.1371/journal.pone.0159825.g003

In regard to SSR motif length, dinucleotide (31.42% of total) and tetranucleotide (25.58% of total) repeats were the most abundant. While, hepta- and octa-nucleotide repeats were the least common, representing together less than 3% of total SSRs (Fig 2, S4 Table). SSR frequency decreased as the motif size increased. The mean number of dinucleotide repeats was 8.48, almost twice as many as trinucleotides and three times as many as tetra- to octanucleotides (S4 Table).

Regarding the distribution of identified SSRs according to the motif type and number of repeats, three-repeat tetranucleotides were the most abundant SSR motif (3424), which was 21.47% of identified perfect SSRs, followed by four-repeat trinucleotides (14.07%) (S4 Table). Di- and trinucleotides with fewer than four repeats were not included in this analysis. The largest SSR identified was the AC dinucleotide with 89 bp, followed by the AGT trinucleotide with 27 bp (Table 4).The most common SSR was AG/CT, with 28.4% of total perfect SSRs, followed by ATT/AAT (Table 4).

Development of SSR genomic markers.

Primer pairs were designed and synthesized for a total of 50 non-redundant sequence SSRs (S2 Table). All 50 primer pairs were screened for amplification of DNA in standard conditions from a mix of ten maqui genotypes. This analysis identified a set of 44 markers (90%) that amplified expected size amplicons. However, only a subset of 11 SSR markers showed scorable polymorphic bands (Table 5). The remaining 39 SSR loci showed no amplification, multibanding patterns, or monomorphic bands. Thus, with these 11 SSR markers we genotyped 40 maqui accessions, including 17 from Río Los Cipreses National Reserve and 23 other locations in Chile (Fig 1, S1 Table).

Table 5. Characteristics of 11 nuclear microsatellite loci developed for maqui (A. chilensis).

For each locus, the forward and reverse primer sequences, repeat motif and annealing temperature when run individually (Ta) are shown.

https://doi.org/10.1371/journal.pone.0159825.t005

Allelic data obtained from 40 genotypes were used to calculate the polymorphism information content (PIC) of each SSR marker, which ranged from 0.117 to 0.820, with an average of 0.584 (Table 6).

Phylogenetic relationship among maqui genotypes from Río Los Cipreses Reserve and other locations in Chile

Fig 4 shows the phylogenetic tree of the 40 maqui genotypes. Three main branches were observed, but they were not supported by significant bootstrap values, suggesting a panmictic structure of maqui. However the 40 genotypes tend to be grouped according to their geographic location. One branch is composed of samples from San Cristobal hill (Metropolitana Region) to Osorno (Los Lagos Region), including a sample from Río Los Cipreses National Reserve. Another branch is composed of two samples from Talca (Maule Region) and the majority of samples from Río Los Cipreses National Reserve with the exception of samples 50, 62 92 and 98, which tend to be grouped in other branch.

Fig 4. Neighbor-joining tree of maqui (A. chilensis) genotypes.

Forty maqui genotypes were analyzed based on 11 SSR locus variations using DARwin V6. 0.012 software and bootstrap analysis of 1000. Numbers indicate genotypes from Río Los Cipreses National Reserve and letters indicate other locations in Chile where the samples were collected, T: Talca, SC: San Cristóbal Hill, ROM: Romeral, SR: San Rosendo, O: Osorno, M: Mantilhue, P: Pucón, BM: Bahía Mansa and LA: Los Ángeles. I, II and III, correspond to branches I, II and III, respectively.

https://doi.org/10.1371/journal.pone.0159825.g004

Prediction of putative maqui genes

The contig and singleton maqui genomic sequences were analyzed by AUGUSTUS software [45] to predict 11150 putative genes using A. thaliana as a model organism (S6 Table). For functional annotation, the potential coding regions were analyzed by BLAST2GO [46], leading to consistent gene annotations, gene names, gene products and Gene Ontology (GO) numbers. The functional search identified 6115 homologous sequences on the non-redundant custom database.

Gene Ontology allows for categorizing gene products according to three ontologies: molecular function, biological process and cellular component. The search for homologous sequences found 6115 sequences on the non-redundant database. Under molecular function ontology, a large proportion of genes were assigned to two categories: binding (47%) and catalytic activities (41%) (Fig 5A), while under biological process ontology the majority of genes were categorized into metabolic (25%), cellular (21%) and single organism processes (Fig 5B).

Fig 5. Gene Ontology classification of the predicted maqui (A. chilensis) ORFs.

The classification was predicted according to molecular function (A) and biological process (B) using BLAST2GO with an E-6 cutoff.

https://doi.org/10.1371/journal.pone.0159825.g005

Prediction of microRNAs in maqui (A. chilensis)

MicroRNAs are short RNA species that act as key post-transcriptional regulators [51]. We used the HHMMIR approach [47] to predict 9229 pre-miRNAs, which were subsequently reduced to 1887 putative candidate miRNAs using miPred and miRdup tools [48–49]. We then searched for regions of local similarity among candidate miRNA sequences using the Basic Local Alignment Search Tool (BLAST) against different plant species to find conserved putative miRNAs.

Three candidate miRNAs were homologous with sequences in miRbase databases, namely miR156, miR812 and miR5149, whose target genes are squamosa-promoters like protein (SPL), ATP binding protein and RPS2 protein, respectively [52–54] (Table 7).

Discussion

This study provides the first set of nuclear microsatellite (SSR) loci for maqui (A. chilensis) by next generation sequencing (NGS). We used 454 pyrosequencing data to obtain a large number (15950) of potential microsatellite sequences. Using a population of 40 maqui genotypes collected mainly at Río Los Cipreses National Reserve (O’Higgins Region, Chile) we characterized 50 SSR. A subset of 11 polymorphic SSR loci that presented good scoring quality was selected for further genetic studies. These microsatellite loci will be useful to investigate genetic structure and gene flow and for developing maqui breeding strategies. This is the first time that part of maqui genome has been made known, which is important in order to learn more about this species with potential as an antioxidant, natural dye and food source [1–3].

Recent developments in sequencing technologies and bioinformatic analysis provide an unprecedented opportunity to discover SSR markers of high quality and effective cost/time in non-model organisms, like maqui, about which genomic information was lacking [35,37,55]. De novo 454 pyrosequencing covered around 64 Mb of the maqui genome, with 165043 reads and 64,018,929 bases (Table 2). An average of 38.94% of GC content of maqui genomic DNA was generated in this study, which is consistent with reports on GC content in other plant genomes like arabidopsis (36%), cucumber (32.3%) grape (34.4%), rice (43.6%) and potato (34.8%) [55,56].

We predicted 11150 putative maqui genes with this information and obtained partial sequences. Ontological analysis of molecular function showed a large proportion classified in binding (47%) and catalytic activities (41%) (Fig 5A), while the ontology of biological processes revealed large proportions in metabolic and cellular processes (Fig 5B). Although these results represent a partial sequencing of the maqui genome, they are the first global identification of maqui genes (S6 Table). Interesting, we also found 14 putative anthocyanidin-related genes from maqui sequences (S7 Table). Among them are putative structural genes like phenylalanine ammonia-lyase and chalcone synthase and some putative regulator genes.

We identified three microRNAs (Table 7), which are short RNA species derived from hairpin-forming miRNA precursors (pre-miRNA) and acting as key post-transcriptional regulators [51]. Most computational tools labeled as miRNA predictors are in fact pre-miRNA predictors and provide no information about the putative miRNA location in the pre-miRNA [47–49]. The identified miRNAs were miR156, miR812 and miR5149, which appear to be related to vegetative phase change, cold stress, and plant disease resistance, respectively [57–59].

Data from 454 sequencing were used to obtain a total of 25494 putative SSR of maqui, where 15950 were classified as perfect microsatellites (Table 3, S3 Table). We detected a density of 382.72 SSR markers per Mb (or 1 SSR each 2.61 kb), considering the total of putative SSR. A higher density of SSRs is described in plants as cucumber [55] but a less density is observed in pigeonpea, among others [60].

The identified microsatellites were genomic SSRs. The development of genic SSRs, SSRs from transcriptomic sequences, is limited to species for which sufficient sequence data are available [61]. Genomic SSRs are better than genic SSRs for fingerprinting or varietal identification studies because greater DNA sequence conservation in transcribed regions in genic SSRs results in less polymorphism, making them less useful than genomic SSRs for distinguishing closely related genotypes [62].

In this study, we analyzed the distribution and frequency of perfect microsatellites with a range of 2 to 8 SSRs and a length >12 bp. The perfect SSR markers are DNA sequences in which a fragment is repeated consecutively without interruption. We used only perfect SSRs because they have greater genetic variation than imperfect SSRs [27]. We considered dinucleotide repeats of ≥ 6, trinucleotide repeats of ≥4, and tetra-, penta-, hexa-, hepta- and octanucleotide repeats of ≥3 (Tables 3 and 4), while mono-nucleotide repeats were excluded from the analysis. The largest percentage (31%) of the 15950 perfect SSRs were dinucleotide sequences (Fig 2), as is the case with the European alder (Alnus glutinosa (L.) Gaertn.), cranberry (Vaccinium macrocarpon Ait.) mung bean (Vigna radiata (L.) Wilczek) and others [37,63–66]. The number of motifs of all types of SSRs decreased as the number of repeats increased. The SSR markers with two tandem repeats had the highest frequency (31, 42%), followed by SSR markers with four tandem repeats (25, 58%) and three tandem repeats (24, 10%) (Fig 2 and S4 Table).

The most common dinucleotide SSR is AG/CT, with a frequency of 28 (4%). With 89pb, AC is the longest SSR (Table 5). In general, AT-rich repeats prevail in dicot species, but not in monocots [55]. In our case, this result could be because is a partial sequencing of maqui genome. AT-rich repeats tend to predominate in trinucleotide SSRs in different plants [55], as was the case in our study, where ATT/AAT was the most common trinucleotide (Table 5).

Based on the identified SSR sequences, fifty pairs of primers were synthetized to evaluate polymorphic SSR loci. Once the polymorphic SSR loci were established, some were used to evaluate their variability in maqui genotypes. Based on 11 polymorphic SSR loci, the phylogenetic tree of the 40 maqui genotypes tend to be grouped into three branches, but they were not supported by significant bootstrap values. This is suggesting a panmictic genetic structure of maqui. This could be due to gene flow throughout the species’ growth distribution and seed dispersion by birds [67,68]. Two branches represent 90% of the samples. One branch is composed of samples from San Cristobal Hill (Metropolitana Region) to Osorno (Los Lagos Region). Another branch is composed by two samples from Talca (Maule Region) and the samples from Río Los Cipreses National Reserve, with the exception of samples number 50, 62, 92 and 98 (Fig 4). Because Río Los Cipreses National Reserve was previously managed as a farm (CONAF staff and A. Lara, Personal Communication) and it suffered big fires before to become a protected reserve, most maqui genotypes come from new seeds (dispersed by birds) from surrounding areas. This fact might explain the low differentiation of its maqui population with the rest of the country. Further studies with bigger sizes of populations and higher numbers of SSRs are necessary answer this type of questions.

Previously, a study differentiated four sampling sites/geographic regions using a fingerprinting approach with inter simple sequence repeats (ISSRs) [67]. However, some maqui genotypes were included in clusters not associated with their geographic origins. The ISSR multi-loci technique has certain disadvantages like the possible non-homology of fragments of similar size and reproducibility problems like RAPDs [69].

Genome-wide analysis of SSRs, coupled with information on their distribution in coding and non-coding regions, can provide insights into the role of SSRs in gene regulation and genome organization [55]. This will be easier to achieve with maqui once its genome has been elucidated.

In this study we used next generation sequencing to identify partial gene sequences, some miRNAs and a large set of SSR markers for first time in maqui (A. chilensis). This information is an important resource for genetic, genomic and evolutionary studies and will aid maqui conservation and breeding programs.

Acknowledgments

Thanks to CONAF and the staff of the Río Los Cipreses National Reserve, as well as A. Lara and R. Contreras for help in sampling.

Author Contributions

Conceived and designed the experiments: AB BS CM. Performed the experiments: AB FC RA PR. Analyzed the data: FC AB BS. Contributed reagents/materials/analysis tools: AB BS RA. Wrote the paper: AB FC PR RA CM BS.

References

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  3. 3. Alonso JR (2012) Maqui (Aristotelia chilensis): a Chilean nutraceutical of medicinal relevance. Rev Farmacol Chile 5: 95–100.
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  5. 5. Muñoz O, Christen P, Cretton S, Backhouse N, Torres V, et al. (2011) Chemical study and anti-inflammatory, analgesic and antioxidant activities of the leaves of Aristotelia chilensis

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