Deep sequencing of the Mexican avocado transcriptome, an ancient angiosperm with a high content of fatty acids
© Ibarra-Laclette et al. 2016
Received: 19 January 2015
Accepted: 14 July 2015
Published: 13 August 2015
Avocado (Persea americana) is an economically important tropical fruit considered to be a good source of fatty acids. Despite its importance, the molecular and cellular characterization of biochemical and developmental processes in avocado is limited due to the lack of transcriptome and genomic information.
The transcriptomes of seeds, roots, stems, leaves, aerial buds and flowers were determined using different sequencing platforms. Additionally, the transcriptomes of three different stages of fruit ripening (pre-climacteric, climacteric and post-climacteric) were also analyzed. The analysis of the RNAseqatlas presented here reveals strong differences in gene expression patterns between different organs, especially between root and flower, but also reveals similarities among the gene expression patterns in other organs, such as stem, leaves and aerial buds (vegetative organs) or seed and fruit (storage organs). Important regulators, functional categories, and differentially expressed genes involved in avocado fruit ripening were identified. Additionally, to demonstrate the utility of the avocado gene expression atlas, we investigated the expression patterns of genes implicated in fatty acid metabolism and fruit ripening.
A description of transcriptomic changes occurring during fruit ripening was obtained in Mexican avocado, contributing to a dynamic view of the expression patterns of genes involved in fatty acid biosynthesis and the fruit ripening process.
Avocado (Persea americana Mill.) is a crop plant with oleaginous fruits belonging to the Magnoliidae clade, a basal lineage of flowering plants. It is a member of Lauraceae, a large family of about 50 genera and approximately 2500–3000 species, mostly trees [1, 2]. Despite its recent introduction to international commerce, the avocado is no longer just an exotic fruit; it has been rapidly incorporated as a key component in the diet of many countries . Although Mexico is the world’s largest producer and consumer of avocados (about 28 % of total world production), there are at least ten other countries with annual production of over 100,000 t of avocado fruit (FAOSTAT, 2011;http://faostat.fao.org/DesktopDefault.aspx?PageID=339&lang=es).
P. americana comprises no fewer than three well-recognized varieties, geographical ecotypes, or botanical races, also known as horticultural races. The Mexican race, P. americana var. drymifolia (Mexican avocado), is adapted to the tropical highlands and constitutes the most commonly used rootstock in Mexican orchards; P. americana var. guatemalensis (L.O. Williams), the Guatemalan race, which grows preferentially at medium elevations in the tropics, and the West-Indian race, P. americana var. americana, which is typically cultivated in the lowland humid tropics . Commercial avocado production is based on grafting cultivars onto rootstocks of Mexican and Guatemalan races; the cultivars grown in subtropical climates are selections from these races or hybrids of them, with the Guatemalan genotypes being the dominant horticultural race among subtropical avocado cultivars . However, the Mexican genetic background contributes to avocado diversity with a plethora of desirable, selected characteristics in commercial varieties, such as cold tolerance, smaller tree size, high oil content, early maturity, and smooth fruit skin. It is imperative to identify key genes and the signaling pathways associated with these traits, as well as to study the allelic diversity present among botanical races for these desirable traits.
The avocado fruit accumulates oil instead of sugar unlike most fruits, probably as a consequence of co-evolutionary processes developed with ancient neotropical megafauna that became extinct about 30,000–11,000 years ago . Avocado has been described as the most nutritious of all fruits , as the mature fruit flesh of avocado contains about 20 % beneficial fatty acids, 6 % carbohydrates, 2 % protein, and vitamin precursors and antioxidants such as carotenoids and vitamins E, C, B2, B12, B1, K and D . The mesocarp of the Mexican race avocado possesses up to 25–30 % oil content, of which nearly 90 % is mono-unsaturated oleic, palmitic and linoleic fatty acids. Although avocado is a strongly climacteric fruit, its ripening or softening process does not take place during maturation on the tree, but instead it starts several days after the fruit has been picked. In the avocado fruit oil content increases in the mesocarp a few weeks after the fruit sets, and healthy fruits on trees continue to grow and accumulate oil for several months after maturation . Once an avocado fruit has been detached from the tree, an ethylene-dependent ripening process is triggered, orchestrating flesh softening, skin color change and lipid biosynthesis. Upon ripening completion, concentrations of unsaturated fatty acids increase and those of saturated fatty acids decrease .
The production of essential oils in avocado vegetative organs is influenced by environmental and developmental conditions. However, the distinctive characteristics of the avocado fruit, including secondary metabolite production, are genotype-dependent. For example, the chemical composition of the leaves of the Mexican race is distinctive in its anise scent, which is absent from the two other horticultural races. Estraole, which represents 60 % of the total essential oils in leaves of the Mexican race, is responsible for this trait [9, 10]. Essential oils contain a variety of volatile molecules such as terpenes and terpenoids, phenol-derived aromatic components, and aliphatic compounds; these are widely used for various pharmaceutical, sanitary, cosmetic, agricultural and food purposes, and in nature, they function as herbivore repellants and pollinator attractants [10, 11]. Understanding the biosynthetic pathways of such bioactive compounds by associating functional genomics information with the enzymes involved in the metabolic pathways is fundamental for their commercial production.
Because to date there are only limited avocado genomic resources, carrying out comprehensive gene expression profiling is a challenge requiring large-scale analysis of transcriptomic data. Breakthroughs in next generation sequencing technology and data analysis during the last 10 years have made it possible to generate reference transcriptomes in the absence of a reference genome at a relatively low cost . Reference transcriptomes can be used to perform comparative expression profiling by methods such as digital gene expression profiling . Here, we present the de novo assembly of the Mexican avocado (Persea americana var. drymifolia) transcriptome, based on hybrid sequencing datasets derived from GS-FLX+ Roche and MiSeq Illumina platforms. Additionally, using a high-throughput sequencing platform, we develop a gene expression atlas of the avocado transcriptome in which a total of six different avocado organs and three fruit ripening stages (pre-climacteric, climacteric and post-climacteric) were included. To confirm the utility of the avocado transcriptome atlas, we specifically analyzed the expression of genes involved in acyl-lipid metabolism, ripening processes, and organ-specificity. Our approach generated over 67,000 unigenes with high quality annotations, providing an unprecedented coverage of the avocado transcriptome. The availability of the avocado gene expression atlas should facilitate additional studies on the basic biology of avocado, while also supporting applied research to improve this increasingly important crop.
Results and discussion
Sequencing and assembly of the Mexican race avocado transcriptome
Reads generated by GS-FLX+ were masked using the SeqClean software pipeline to eliminate sequence regions that would cause incorrect assembly while the MiSeq read pairs (2 × 150 bp) were trimmed and/or merged together using the SeqPrep pipeline (see Methods for more details). To carry out the assembly process, 4,530,278 high quality reads (931,834 generated by GS-FLX+ and 3,598,444 paired-reads generated by MiSeq) were considered (Additional file 1: Table S1).
Many assemblers have been developed to assemble reads generated by Next Generation Sequencing platforms (NGS). The overlap layout algorithm is able to handle the longer reads of GS-FLX+, and programs such as MIRA  incorporate it. Trinity  in contrast is a de-Bruijn graph-based assembler developed for short reads. We compared the performance of the MIRA v3.4.1 and Trinity assemblers, both previously used in several analyses of plant transcriptomes [17, 18]. For each assembler, we used the default parameters recommended for transcriptome assembly. Standard metrics describing the assembly process, such as number of contigs ≥1 Kb, average contig lengths and maximum contig size were used to compare the assembly programs (Additional file 1: Table S2). Considering that many contigs representative of unique genes are often produced in de novo assemblies due to the presence of variant alleles, sequencing errors, and alternative splicing of transcripts, the resulting contigs (derived from both assemblies) were filtered to eliminate redundant sequences and then passed through a second assembly step using the CAP3 assembler  (see Methods for more details). A unigene set (83,650 sequences) from P. americana was generated including resulting contigs (25,665) and “singlets” (57,985; 64.2 % from MIRA and 33.8 % from Trinity) derived from the CAP3 run with a minimum size of 200 bp (Additional file 2). It should be noted that “singlets” are the contigs generated from the first steps of MIRA or Trinity assemblies that were not reassembled by CAP3. The average length of unigenes was 816.21 bp (ranging from 0.2 to 8.6 kb) (Additional file 1: Table S2). Considering the mean size of coding sequences (≈942.16 bp) in Amborella trichopoda, a basal angiosperm species [20, 21], it was expected that a large percentage of these avocado unigene transcripts may represent full-length cDNAs. A comparison of P. americana unigenes against the unpublished ca. 800 Mbp draft genome of P. americana var. drymifolia (unpublished data) using BLASTN (e-value 10−3) shows that 94.65 % of the transcripts had a significant hit against the genome (98 % of alignment length and minimal sequence identity of 90 % over the complete alignment).
To annotate the avocado transcriptome, we performed BLASTX alignments (e-value of ≤10−03 and a bit score ≥25) between the unigene set and several protein databases, including Arabidopsis thaliana, Amborella trichopoda and plant proteins available in the Reference Sequences (RefSeq) collection of NCBI. We found that 67,709 (80.94 %) unigenes of P. americana show high identity to at least one plant protein; the remaining (15,941 unigenes) had no function assigned (Additional file 1: Table S3). In a total of 14,845 avocado unigenes, an individual high-scoring segment pair (HSP) produced by BLASTX covered at least 80 % of the target protein. Results indicated that 34,218 distinct plant proteins could be identified among the 63,459 unigenes that showed significant similarities against RefSeq database (Additional file 1: Table S3). We further compared P. americana unigenes against the Pfam (Protein families) domain database (Additional file 1: Table S3; see Methods for more details) .
Expression map of the P. americana unigenes (the transcriptome atlas)
In the past, gene expression atlases from different plant species have been established by using massive parallel-signature sequencing and array-hybridization technologies [25–30]. Plant transcriptomes strongly vary from one tissue to another [25, 29], and it has been suggested that these variations are responsible, at least in part, for the identities of different plant organs . We used the SOLiD v4.0-sequencing platform to quantify the expression of P. americana unigenes in seven different organs: seeds, roots, stem, leaves, aerial buds, flowers and fruits. Fruit was considered as the total of reads generated from pre-climacteric, climacteric and post-climacteric libraries. The resulting reads from these three libraries were also independently mapped. Between 4.18 and 6.84 million SOLiD reads were generated for each of the seven organ libraries; among them, 51.13 % were mapped to the avocado transcriptome (Additional file 4: Table S5). An expression profile matrix containing the unigenes (rows) and the number of mapped reads in each normalized organ-specific transcriptome (columns), was created. To allow for data comparison among samples, a normalization of the reads per Kb per million (RPKM) was performed. A threshold of RPKM ≥5 was used to distinguish expressed genes in at least one of the organs sampled from background  (Additional file 4: Table S6). We further investigated how the organs can be classified according to transcript levels using a principal component analysis (PCA) and hierarchical clustering (HC), by employing the Pearson metric on average expression levels for each organ (Fig. 3a and b).
For many of the organ types, the hierarchical plot and the PCA reflect the known similarity of biological functions among organs. For example, transcripts from leaves and stems clustered as a neighboring group, reflecting their physiological similarity (vegetative organs). Likewise, the storage organ transcriptomes (fruit and seed) were grouped together, whereas gene products from roots clustered separately from the rest (Fig. 3b). To assess the relative abundance of gene transcripts among the organ-specific transcriptomes, we used the log-likelihood ratio statistic, R , which scores reads by departures from the null hypothesis of equal counts in each library given the total number of reads sampled from each library. Higher R-values indicate a greater probability of differential expression, whereas R-values near zero represent constitutive expression (ubiquitous unigenes; see Methods). By considering as preferentially expressed genes the unigenes with R-values ≥15 (true positive rate of ~98 %), a total of 9357 P. americana unigenes were selected as preferentially expressed in at least one of the organ analyzed (Additional file 4: Table S7). Of these, 1968 unigenes (224 from seed, 192 from leaves, 22 from stem, 503 from roots, 56 from aerial buds, 654 from flowers and 313 from fruit) could be considered as organ-specific genes because the reads were derived from a single library (Additional file 4: Table S8). Although 33.63 % of organ-specific unigenes corresponded to sequences that could not be annotated, in the remaining unigenes, these organ-specific expression data are consistent with previously documented expression patterns of known genes. For example, the avocado unigene UN06501, a homolog of the Arabidopsis high-affinity phosphate transporter PHT1-3 gene (AT5G43360), is expressed in roots . Four well-characterized floral pattern determination genes, AGL2 (AT5G15800; homologous to UN50397), AGL6 (AT2G45650; homologous to UN22054), AP3 (AT3G54340; homologous to UN19620) and PI (AT5G20240; homologous to UN19579), exhibited specific expression patterns among floral organs [34–37]. Likewise, in leaves, several avocado unigenes homologous to Arabidopsis light-harvesting chlorophyll a/b (LHCB) proteins were expressed in an organ-specific manner. This was expected, since in higher plants and algae, LHCB proteins are major components of the light-harvesting complex of photosystem II (PSII) in chloroplasts, which is responsible for light harvesting and energy transfer to reaction centers [38, 39]. Moreover, the fruit-specific avocado unigene UN44288 is homologous to AGL4 (AT3G02310), a transcription factor that has been recently characterized for its main role in apple fruit development and ripening . Interestingly, an oleosin-encoding gene (UN35609) homologous to AT3G18570 showed a higher expression level in fruits than in seeds (Additional file 4: Table S7). Oleosins have been described as key structural and signaling components of seed oil bodies that form during the desiccation process and prevent coalescence of the oil . This suggests that oleosins could be involved in avocado fruit development, beyond their known functions in seeds. In addition to its high lipid content, the avocado fruit contain several bioactive phytochemicals, including carotenoids . The homolog of carotenoid biosynthesis-associated epoxycarotenoid dioxygenase NCED3 (AT3G14440), unigene UN43474, was highly expressed in the fruit transcriptome (Additional file 4: Table S7). Seven different avocado unigenes (UN26753, UN45556, UN14598, UN31059, UN39923, UN51437 and UN56049), among the highest expression levels from seed-specific genes, are homologs of PAP85 (AT3G22640), a member of the large superfamily of cupins, which are expressed during seed development and act as a nutrient reservoir . The homolog to fasciclin-like arabinogalactan protein FLA12 (AT5G60490; unigene UN17820) exhibited one of the highest expression levels of stem-specific genes, which is consistent with previous reports showing that expression of some members of the FLA gene family is correlated with the onset of secondary-wall cellulose synthesis in Arabidopsis stems and with wood formation in the stems and branches of trees, suggesting a biological role in avocado stem development . Together, these results indicate that the transcriptome atlas of P. americana presented here provides an accurate estimation of organ-specific gene expression patterns that may assist functional interpretations.
Transcriptome changes during avocado fruit development and ripening
We identified unigenes that are differentially expressed during fruit development and ripening using a similar approach to that described above (RPKM values derived from mapped reads and a threshold of R ≥15 to select differentially expressed genes; see Methods). First, the RPKM values between flower and fruit were compared in order to identify some genes that may play important roles during fruit development. A total of 382 unigenes were selected due to the significant increase of their transcripts in the fruit/flower comparison. We surveyed some of these genes in order to determine their potential roles during fruit development (Additional file 4: Table S9). A total of 10 avocado unigenes (UN54812, UN37501, UN61103, UN59855, UN53293, UN48116, UN58333, UN64333, UN64163 and UN68295), homologs of Arabidopsis metallothionein MT2A (AT3G09390), were identified. Metallothioneins are small cysteine-rich proteins required for heavy metal tolerance in animals and fungi. In plants, metallothionein genes are up-regulated in response to heavy metal stress  but also participate in natural and induced leaf senescence [46, 47], ethylene-induced abscission  and biotic and abiotic stress responses [49–51]. However, metallothioneins have been also identified as up-regulated genes during climacteric fruit development in banana , apple , and kiwifruit  and in non-climacteric fruit such as grape , Citrus unshiu , strawberry  and pineapple . Despite their abundance, the function of metallothioneins during fruit development still remains largely unknown. Chitinases were also found to be a well-represented group of fruit specific unigenes (18 in total), most of them homologs of the basic chitinase CHIB/PR-3 (AT3G12500). Consistently, besides the role of chitinases in plant defense , these proteins have also been associated with fruit development in both climacteric  and non-climacteric fruits . Pectate lyases constitute an additional protein family that has been suggested to play an important role in fruit ripening and softening . This is consistent with the fact that eight avocado unigenes, homologous to four different members of the Arabidopsis pectin lyase-like superfamily (AT1G80170, AT3G07850, AT3G07970 and AT5G18990), showed significant increase in the frequency of transcriptional units in the flower/fruit comparison.
Genes relevant to avocado fruit ripening
In ripening of climacteric fruits, the expression of some members of the gene families encoding ACC synthase and ACC oxidase is induced, regulating the biosynthesis of ethylene . Two differentially expressed unigenes (UN03798 and UN39755) were found to be homologs of Arabidopsis ACC synthase (AT3G61510), and one, UN38306, a homolog of ACC oxidase (AT1G62380). In relation to their expression profiles, the unigenes homologous to the ACC synthase were classified into class III-A, while the homolog of ACC oxidase was classified into class II (Fig. 5b). Therefore, during avocado fruit ripening, the ethylene burst seems to be regulated mainly by ACC oxidase. This is consistent with the notion that in climacteric fruits the rate of ethylene production is well correlated with the pattern of accumulation of ACC synthase and ACC oxidase gene transcripts . Avocado unigenes homologous to ethylene response factor ERF110 (AT5G50080; UN29560 and UN42714) and ethylene receptor EIN4 (AT3G04580; UN01530 and UN24061), which play key roles in the signaling of ethylene responses , were also identified as members of class III-A (Fig. 5b).
The genetic and physiological characterization of tomato ripening mutants, ripening-inhibitor (rin; ), non-ripening (nor; ) and colorless non-ripening (cnr; ), together with the molecular characterization of the mutated genes, have demonstrated that several important regulatory factors must be properly coordinated with the ethylene signal to properly activate and orchestrate the ripening program. RIN , NOR  and CNR  genes encode transcription factors belonging to the MADS-box, NAC-domain, and SBP-box families, respectively, that act upstream of ethylene biosynthesis and perform key functions in the control of fruit ripening . RIN  and two regulatory proteins more recently identify as involved in fruit ripening, the TAGL1 MADS-box factor  and the HB-1 homeobox protein , are able to bind to the promoter region of ACS2  and ACO1  genes, respectively, demonstrating that transcription factors directly regulate the activity of ethylene biosynthesis genes in tomato. A bi-directional best BLAST hit approach was used to identify the avocado orthologs of tomato genes involved in ripening (see above). Using the SeaView program  the protein-coding nucleotide sequences were then aligned based on their corresponding amino acid translations to calculate the percent identity at nucleotide and amino acid levels (Additional file 5). The identities of avocado/tomato genes ranged from 48.82 to 63.53 % (Additional file 4: Table S12) and with only one exception (HB-1; UN22151), their expression levels were considerably higher in flowers than in the remaining organs sampled. Meanwhile, NOR and HB-1 avocado transcripts increased during fruit ripening while CNR decreased. RIN and TAGL1 showed very low transcript levels (RPKM <5) only at the preclimacteric stage (Additional file 3: Figure S3). Avocado is considered a basal angiosperms with origin near the split between monocot and eudicot plant species. The finding that some but not all transcription factors involved in tomato fruit ripening are up-regulated or even expressed during avocado fruit ripening suggests that part but not all of the transcription factor wiring was ancestrally present and that substantial rewiring occurred during the evolution of modern eudicots.
It has been reported that a thaumatin-like protein and a class I chitinase are some of the most abundant ripening-associated proteins in banana fruit . Genes encoding these proteins were also found to have differential expression during ripening in avocado. Avocado unigene UN10274, a homolog of a pathogenesis-related thaumatin (AT1G20030), clustered into class III-A (transcripts continuously rising; Fig. 5b), the same as several unigenes (16 in total; see Additional file 4: Table S10) homologous to PR-3 (AT3G12500), a well-known class I chitinase strongly induced when plants respond to wounding or infection by fungal, bacterial, or viral pathogens . Additional unigenes (UN43717 and UN45051) with different expression profiles (class I) were identified as homologs of AT4G01700 and AT2G43610, class II and IV chitinases, respectively. Therefore, comparisons with data reported for other climacteric fruits (including ethylene responsive genes) facilitated identification of putative conserved orthologous ripening-related genes, which serve as an initial set of candidates for assessing the conservation of gene activity during the evolution of fleshy-fruited plant species.
Acyl lipid metabolism of Persea americana fruits
Although in most avocado varieties over half of the total fat present is in the form of oleic acid (C18:1), recent studies have shown that at least 22 different fatty acids can be detected in pulp and seed avocado oils . Palmitic (C16:0) and linoleic (C18:2) fatty acids are the second major constituents of fruit oils while some others like stearic acid (C18:0) are present in trace amounts . The benefits to nutrition and health of avocado fatty acids have been recently reviewed ). In addition, avocado acetogenins, such as persin [(+)-(Z,Z)-1-(acetyloxy)-2-hydroxy-12,15-heneicosadien-4-one], a deoxy-derivative of glyceride with close structural homology to the monoglyceride of linoleic acid, has been proposed as an alternative therapy against breast cancer due its necrotic effect in the mammary gland . Considering the importance of avocado to nutrition and health, transcripts encoding enzymes of the biosynthetic pathway from palmitic to linoleic acid, passing through stearic and oleic acids, were reconstructed similarly to described above (Additional file 4: Table S13; Additional file 3: Figure S4). It is worth noting that genes involved in the biosynthesis of linoleic acid (downstream of oleic acid) showed lower expression levels in fruit than in seed. This is consistent with previous reports in which linoleic acid seems to accumulate in greater amounts (around 4-fold, ) in seeds than in fruits.
The avocado transcriptome reported in this study provides a foundation for the molecular genetics and functional genomics required to study the basis of the genetic diversity that determines the different levels and quality of oil accumulation in the fruit of different avocado cultivars, as well as other important agronomic traits for this increasingly important crop. Transcriptomic data will also facilitate the identification of the pathways involved in production of a wide variety of essential nutrients and phytochemicals beneficial for human health that are produced by the avocado. This data will also facilitate the study of early plant evolution since avocado is phylogenetically placed near the separation between monocot and eudicot plants.
Avocado (Persea americana var. drymifolia) samples were obtained from the germplasm bank of the “Instituto Nacional de Investigaciones Forestales y Agropecuarias (INIFAP)” in Uruapan, Michoacán, México. Organs sampled [Leaves: mixed development stages of expanded leaves; Stem: segments from young branches, Aerial buds: developing buds emerging from shoot apical meristem; Flower: whole inflorescences with flowers at multiple stages of development (young, immature, and mature); Seed: isolated from harvested fruits at the mature-green state (approximately 8 months-old); roots: whole root system recovered from in vitro-propagated seedlings , or from cuttings grown in pots in a greenhouse]. Fruits in the mature-green state were also harvested for the ripening experiments. Seeds were removed once the fruit was frozen at each rippening stage. All organs/tissues sampled were obtained from a single reference tree (accession 001-01, which has been sequenced as part of the avocado whole-genome sequencing project).
Characterization of the climacteric behavior of avocado fruit
In avocado and other climacteric fruits such as apple, melon and banana, an ethylene burst is required for normal fruit ripening (reviewed in ). According to the method described by Hoffman and Yang , ethylene production of each fruit was monitored individually by gas chromatography during the course of ripening until they were sacrificed for RNA extraction.
Total RNA was isolated using the Trizol reagment (Invitrogen) and re-purified with the RNeasy kit (Qiagen) following the manufacturer’s instructions. Five micrograms of total RNA from each sample were pooled to produce sequencing libraries. Additionally, according to ethylene quantification (Additional file 3: Figure S1), three independent extractions were prepared from 8 months-old fruits (pericarp), at the second, third and fourth day after harvest (pre-climacteric, climacteric and post-climacteric stages respectively), allowing us to contrast changes in the transcriptome profile of the avocado fruit at different ripening stages. cDNA preparation, library construction, and sequencing were performed according to Illumina and Roche manufacturer instructions. From the RNA pool, two sequencing libraries were prepared and a single sequencing run was carried out on the GS-FLX+ (Roche) sequencer, and another on the Illumina MiSeq platform. A total of 4,674,756 reads were generated (1,055,903 reads from GS-FLX+ with an estimated average size of 462.27 bp and 3,618,853 paired reads, 2 × 150 bp, from MiSeq). Additionally, using the SOLiD system and the SOLiD RNA Barcoding Kit (v4 chemistry), 1/4 run, in which all RNA samples were independently represented using barcodes, was performed. A total of 70,650,328 short reads (up to 50 pb) were generated (Additional file 1: Table S1). Files containing sequence reads and quality scores were deposited in the Short Read Archive of the National Center for Biotechnology Information (NCBI) [Accession number SRS923862].
Assembly and sequence analysis
To carry out the assembly process only the reads generated with GS-FLX+ and Illumina-MiSeq sequencer were considered. Using the CDHIT program , natural and artificial duplicate reads were removed from the data set generated by the GS-FLX+ sequencer. Additionally, reads with an average quality less than 20 (phred score), were also removed. On the other hand, forward and reverse read pairs (generated by Illumina-MiSeq) were merged to form single “longer-reads” using the SeqPrep pipeline (https://github.com/jstjohn/SeqPrep), with default parameters (a quality score cutoff of phred 33, a minimum merged read length of 15 bp and no mismatches in the overlapping region). Paired-end reads that did not overlap were trimmed using a sliding window approach (window size 10 bases, shift 1 base). Reads were discarded if they were smaller than 30 bases after trimming, and orphan reads were also removed in order to keep pairs only. MIRA v3.4.1  and Trinity  assemblers were used independently. Standard assembly metrics, such as number of contigs ≥1 Kb, average contig lengths, and maximum contig size were estimated (Additional file 1: Table S2). Resulting contigs (with a minimum length of 100 bp) derived from both assembly processes were merged into a single file, and those that were redundant were eliminated using the BlastClust program. Redundant contigs were defined as those having greater than 95 % identity over an area covering 95 % of the length of the sequence. Unique contigs were trimmed of low quality, low complexity and poly(A/T) tails using the SeqClean software (http://compbio.dfci.harvard.edu/tgi/), then, they were passed through a second assembly step using the CAP3 assembler . CAP3 was run with default parameters (minimum overlap length of 40 bp and 95 % minimum sequence identity). A set of unigenes from P. americana were generated considering only resulting contigs with a minimum size of 200 bp (Additional file 1: Table S2). SOLiD sequence reads were only used for differential expression analysis and they were excluded from the assembly because direct conversion of sequencing reads is possible but not recommended because all bases that follow a single error in colorspace will create errors in all subsequent bases of a read. Additionally, it has been recently reported that a hybrid strategy generated high quality assemblies following three simple recommendations: (1) using a single individual with large representation of biological tissues, (2) merging both long reads and paired-end reads (derived from GS-FLX+ and Illumina platforms, respectively) and (3), using several assemblers in order to combine specific advantage of each .
Annotation of Persea americana var. drymifolia unigenes
To annotate sequences obtained by de novo assembly, we performed sequence similarity searches using the BLASTX algorithm (e-value 10−3, bit score ≥25) on Arabidopsis thaliana (TAIR v11; http://www.arabidopsis.org/) and Amborella trichopoda (http://www.amborella.org/) proteins, and plant proteins from other species available in the Reference Sequence (RefSeq) collection (NCBI; ftp://ftp.ncbi.nlm.nih.gov/refseq/release/plant/). Top protein matches from Arabidopsis, Amborella or other plant species were assigned to each of the avocado unigenes (Additional file 1: Table S3). The putative protein domains contained within the translated unigenes were identified using Hidden Markov Model (HMM)-based searches against Pfam database (e-value 10−3) . The gene ontology (GO) functional classes and pathways for each avocado unigene were assigned based on Arabidopsis GO SLIM and pathway annotation (Additional file 1: Table S3). Association of unigenes with KEGG pathways was determined using a directional best hit (BBH) method (that is, top reciprocal BLAST hits) against the Kyoto Encyclopedia of Genes and Genomes database . The KEGG pathways annotation (Additional file 3) was performed in the KEGG Automatic Annotation Server (KAAS) (http:/www.genome.jp/tools/kaas/) .
Expression profile analysis of the P. americana transcriptome
After assembling the P. americana transcriptome, reads generated by the SOLiD system were separated according to the barcode used, and then were separately aligned to the avocado unigene set using BioScope (Life Technologies). To run whole transcriptome analysis with BioScope, a reference genome is required. Using a custom Perl script, a reference genome proxy was created linking all unigenes derived from the assembly process (in which unigenes were separated by 50 N’s); at the same time, a GTF/GFF file was also created. The libraries were relatively uniform with respect to mapping efficiency (Additional file 4: Table S5). Gene expression levels were calculated by the RPKM (reads per kb per million reads) method, and preferentially expressed genes were selected according to the method described by Stekel . Briefly, all unigenes were submitted to a log-likelihood ratio statistic that trends asymptotically to a χ2 distribution, as described by Stekel et al. . We considered as preferentially expressed those unigenes with a value of R ≥15 among organs sampled (Additional file 3: Figure S5). This provides a single statistical test to describe the extent to which a gene is differentially expressed between libraries. This method permits identification of differentially expressed genes among any number of libraries. Hierarchical clustering was performed using the Pearson correlation coefficient and average linkage clustering . Results were visualized using GeneSpring GX 7.3.1 software (Agilent Technologies).
Real-time quantitative PCR verification
Eight avocado unigenes identified as differentially expressed genes were aligned against their Arabidopsis homologues in order to identify the coding sequences in their correct open reading frame. Protein-coding nucleotide sequences were aligned based on their corresponding amino acid translations using the SeaView program (Additional file 4: Table S14). Gene-specific primer pairs (Additional file 4: Table S15), which were designed using the Primer3 v.0.4.0 web tool (http://bioinfo.ut.ee/primer3-0.4.0/primer3/), were used for real-time PCR.
A total of 10 μg of RNA was reverse transcribed for first-strand cDNA synthesis using SuperScript® III Reverse Transcriptase (Life Technologies) according to the manufacturer’s instructions. Reactions were performed with the SYBR Green PCR Master Mix in an ABI 7500 Fast Real-time system. Actin was used as the standard to normalize the content of cDNA, as described previously . The thermal cycling program was set to 95 °C for 5 min, 40 cycles of 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 1 min. Results were analyzed using the ABI 7500 on-board software, version 2.0.5 (Applied Biosystems). The real-time PCR was conducted with at least three experimental replicates for each biological sample.
Availability of supporting data
All the supporting data are included as additional files, including the sequences of all assembled transcripts. The raw sequencing data was deposited in the NCBI and can be accesed under the following identifier: BioProject PRJNA282441.
This work was funded by SAGARPA, Grant 2009-C02-126261 (LH-E). We thank Flor Zamudio-Hernández and María de Jesus Ortega-Estrada for their technical support in the real-time PCR services, as well as INIFAP authorities for their permission to access the germoplasm bank. We also thank Alexandro Alonso-Sánchez and Emanuel Villafán de la Torre for discussion and critical comments on the manuscript, Araceli Fernández-Cortes who has responsibility for the high-performance computing system at LANGEBIO. Special thank goes to the staff of “Laboratorio de Servicios Genómicos” at LANBEBIO for their assistance with RNAseq library preparation and sequencing services. We thank Le-Shin Wu and the National Center for Genome Analysis Support at Indiana University for computing assistance. We also thank the anonymous reviewers for their positive and relevant comments, which have improved the quality of this manuscript.
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