Identification and Characterization of Microsatellite Loci in Maqui (Aristotelia chilensis [Molina] Stunz) Using Next-Generation Sequencing (NGS)

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.Alto Cachapoal Scientific Research Fund (Pacific Hydro Chacayes, Chile) Fondecyt 112111

Aging gene pathway of microRNAs 156/157 and 172 is altered in juvenile and adult plants from in vitro propagated Prunus sp.

A. Bastías, R. Almada, P. Rojas, J.M. Donoso, P. Hinrichsen, and B. Sagredo. 2016. Aging gene pathway of microRNAs 156/157 and 172 is altered in juvenile and adult plants from in vitro propagated Prunus sp. Cien. Inv. Agr. 43(3):429-441. In vitro culture is a very popular technique to mass propagate valuable plant genotypes, including Prunus sp. cultivars. Plants that undergo tissue culture processes often change their morphology and behavior due to the "rejuvenation" caused by the plant growth regulators included in the medium. To evaluate the effects of rejuvenation by tissue culture in Prunus sp., the expression patterns of the aging gene pathway described in plant models, which include the highly conserved microRNA (miRNA or miR) 156/157 and 172 families and several of their respective target genes, were analyzed in distinct Prunus sp. genotypes at different phases of maturity, including true seedling and tissue culture micropropagated plants. In genotypes from true seedling plants, the expression of miR156 and miR157 was higher in the leaves of juvenile plants (one year old) than in those of adult plants (six year old). The opposite pattern was observed with miRNA172 expression. Our results suggest that the aging gene pathway is relatively conserved in Prunus and likely plays a key role in vegetative phase change. However, Prunus sp. plants that were rejuvenated and propagated by in vitro methods showed more erratic behavior for miR156 and miR157 and their target genes, suggesting that tissue culture alters the normal control of the aging pathway.A. Bastías, R. Almada, P. Rojas, J.M. Donoso, P. Hinrichsen y B. Sagredo. 2016. La vía génica del envejecimiento de los microARNs 156/157 and 172 es alterada en plantas juveniles y adultas de Prunus sp. que provienen de propagación in vitro. Cien. Inv. Agr. 43(3):429-441. El cultivo in vitro es una técnica muy popular para propagar en masa genotipos de plantas valiosas, incluyendo cultivares de Prunus sp. Las plantas que se someten a procesos de cultivo de tejidos a menudo cambian su morfología y comportamiento debido a la "rejuvenilización" causada por los reguladores de crecimiento de planta incluidos en el medio. Para evaluar el efecto de la rejuvenilización por cultivo de tejido en Prunus sp, los patrones de expresión de la vía génica del envejecimiento descrita en plantas modelos, la cual incluye a las familias altamente conservadas de microRNAs (miR) 156/157 y miR172 y varios de sus genes blancos respectivos, fueron analizados en diferentes genotipos de Prunus sp. en fases de madurez distintas, incluyendo tanto plantas provenientes de semilla verdadera como micropropagadas mediante cultivo de tejidos. En los genotipos de plantas que provienen de semilla verdadera, la expresión de microR156 and miR157 fue más alta en hojas de plantas juveniles que en plantas adultas. El patrón opuesto fue observado con el patrón de expresión de miR172. Nuestros resultados sugieren que la vía génica del envejecimiento es conservada en Prunus y probablemente juega una función en el cambio de fase vegetativo. Sin embargo, plantas de Prunus sp. que fueron rejuvenecidas y propagadas por métodos in vitro mostraron un comportamiento más errático para los mi156 y miR157 y sus genes blancos, sugiriendo que el cultivo de tejidos altera el control normal de la vía del envejecimiento

Modulation of organic acids and sugar content in tomato fruits by an abscisic acid-regulated transcription factor

Growing evidence suggests that the phytohormone abscisic acid (ABA) plays a role in fruit development. ABA signaling components of developmental programs and responses to stress conditions include the group of basic leucine zipper transcriptional activators known as ABA-response element binding factors (AREBs/ABFs). AREB transcription factors mediate ABA-regulated gene expression involved in desiccation tolerance and are expressed mainly in seeds and in vegetative tissues under stress; however, they are also expressed in some fruits such as tomato. In order to get an insight into the role of ABA signaling in fruit development, the expression of two AREB-like factors were investigated during different developmental stages. In addition, tomato transgenic lines that overexpress and downregulate one AREB-like transcription factor, SlAREB1, were used to determine its effect on the levels of some metabolites determining fruit quality. Higher levels of citric acid, malic acid, glutamic acid, glucose and fructose were observed in SlAREB1-overexpressing lines compared with those in antisense suppression lines in red mature fruit pericarp. The higher hexose concentration correlated with increased expression of genes encoding a vacuolar invertase (EC 3.2.1.26) and a sucrose synthase (EC 2.4.1.13). No significant changes were found in ethylene content which agrees with the normal ripening phenotype observed in transgenic fruits. These results suggest that an AREB-mediated ABA signal affects the metabolism of these compounds during the fruit developmental program

The transcription factor AREB1 regulates primary metabolic pathways in tomato fruits

Tomato fruit development is regulated both by the action of plant hormones and by tight genetic control. Recent studies suggest that abscisic acid (ABA) signalling may affect different aspects of fruit maturation. Previously, it was shown that SlAREB1, an ABA-regulated transcription factor involved in stress-induced responses, is expressed in seeds and in fruit tissues in tomato. Here, the role of SlAREB1 in regulating the expression of genes relevant for primary metabolic pathways and affecting the metabolic profile of the fruit was investigated using transgenic tomato lines. Metabolite profiling using gas chromatography–time of flight mass spectrometry (GC-TOF-MS) and non-targeted liquid chromatography–mass spectrometry (LC-MS) was performed on pericarp tissue from fruits harvested at three stages of fruit development. Principal component analysis of the data could distinguish the metabolite profiles of non-transgenic fruits from those that overexpress and down-regulate SlAREB1. Overexpression of SlAREB1 resulted in increased content of organic acids, hexoses, hexose-phosphates, and amino acids in immature green, mature green, and red ripe fruits, and these modifications correlated with the up-regulation of enzyme-encoding genes involved in primary carbohydrate and amino acid metabolism. A non-targeted LC-MS analysis indicated that the composition of secondary metabolites is also affected in transgenic lines. In addition, gene expression data revealed that some genes associated with fruit ripening are also up-regulated in SlAREB1-overexpressing lines compared with wild-type and antisense lines. Taken together, the results suggest that SlAREB1 participates in the regulation of the metabolic programming that takes place during fruit ripening and that may explain part of the role of ABA in fruit development in tomato

Results from homology search of microRNA candidates from maqui (<i>A</i>. <i>chilensis</i>) against the microRNA database.

<p>Results from homology search of microRNA candidates from maqui (<i>A</i>. <i>chilensis</i>) against the microRNA database.</p

Number of maqui (<i>A</i>. <i>chilensis</i>) 454 sequences before and after assembly.

<p>Number of maqui (<i>A</i>. <i>chilensis</i>) 454 sequences before and after assembly.</p

Characterization of SSRs in genomic sequences of maqui (<i>A</i>. <i>chilensis</i>) generated by 454 sequencing.

<p>Characterization of SSRs in genomic sequences of maqui (<i>A</i>. <i>chilensis</i>) generated by 454 sequencing.</p

Results of microsatellite (SSRs) search from maqui (<i>A</i>. <i>chilensis</i>) using MIcroSAtellite identification tool.

<p>Results of microsatellite (SSRs) search from maqui (<i>A</i>. <i>chilensis</i>) using MIcroSAtellite identification tool.</p

Gene Ontology classification of the predicted maqui (<i>A</i>. <i>chilensis</i>) ORFs.

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

Frequency distribution of SSR loci of maqui (<i>A</i>. <i>chilensis</i>) by motif length.

<p>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.</p