Transcriptome Sequencing of Different Avocado Ecotypes: de novo Transcriptome Assembly, Annotation, Identification and Validation of EST-SSR Markers

Abstract

Avocado (Persea americana Mill.) could be considered as an important tropical and subtropical woody oil crop with high economic and nutritional value. Despite the importance of this species, genomic information is currently unavailable for avocado and closely related congeners. In this study, we generated more than 216 million clean reads from different avocado ecotypes using Illumina HiSeq high-throughput sequencing technology. The high-quality reads were assembled into 154,310 unigenes with an average length of 922 bp. A total of 55,558 simple sequence repeat (SSR) loci detected among the 43,270 SSR-containing unigene sequences were used to develop 74,580 expressed sequence tag (EST)-SSR markers. From these markers, a subset of 100 EST-SSR markers was randomly chosen to identify polymorphic EST-SSR markers in 28 avocado accessions. Sixteen EST-SSR markers with moderate to high polymorphism levels were detected, with polymorphism information contents ranging from 0.33 to 0.84 and averaging 0.63. These 16 polymorphic EST-SSRs could clearly and effectively distinguish the 28 avocado accessions. In summary, our study is the first presentation of transcriptome data of different avocado ecotypes and comprehensive study on the development and analysis of a set of EST-SSR markers in avocado. The application of next-generation sequencing techniques for SSR development is a potentially powerful tool for genetic studies.

Single-Molecule Long-Read Sequencing of Avocado Generates Microsatellite Markers for Analyzing the Genetic Diversity in Avocado Germplasm

“…The 100 EST-SSR markers randomly selected for validation in the present study had an amplification rate of 67%, and 30 were determined to be polymorphic. This polymorphism level is generally consistent with that of our previous study [18]. In subsequent analyses of the genetic diversity of these polymorphic EST-SSR markers among 46 avocado accessions, 15 markers produced 4.73 alleles per locus, which was fewer than the 6.13 alleles per locus of Ge [18], the 11.40 alleles per SSR locus of Gross-German and Viruel [17], the 18.8 alleles per SSR locus of Schnell [16], and the 9.75 alleles per SSR locus of Alcaraz and Hormaza [62].…”

“…In this study, 93.82% (71,627 of 76,345) of the nonredundant transcripts were annotated based on similarities with sequences in public databases. Thus, a greater proportion of transcripts were annotated in this study than in previous investigations involving NGS data for various avocado races (49.00%) [18] and for avocado mesocarp samples (57.50%) [55]. We determined that the mean length of the avocado nonredundant transcripts was2330 bp, implying that our sequences were long enough to represent full-length transcripts.…”

“…Transcriptome sequencing is a useful technique for obtaining a large number of transcripts for organisms lacking a reference sequence, at least partly because it is inexpensive and can be completed rapidly [51][52][53]. To date, several short-read next-generation sequencing (NGS) transcriptome databases have been developed for avocado mesocarp samples [54,55] and avocado mixed tissue samples [18,56]. However, both the number and length of the transcript sequences derived from these short-read NGS studies have hamperedtheirapplication ingenetics and molecular biology research [41].…”

“…Specifically, a molecular characterization is required for analyses of the genetic relationships among avocado germplasm. Over the past two decades, studies involving various types of molecular markers have examined the genetic relationships among avocado germplasm [11][12][13][14][15][16][17][18][19][20]. Of the many available DNA markers, simple sequence repeats (SSRs) are commonly used for investigating plant genetics and breeding because they are widely distributed and abundant in plant genomes.…”

Molecular Markers and a Quality Trait Evaluation for Assessing the Genetic Diversity of Avocado Landraces from China

“…We previously generated race-specific markers from the specific length amplified fragment sequencing data of different avocado races to validate the racial origins of accessions with an unknown race [21]. Furthermore, highly polymorphic EST-SSR markers were generated from the transcriptomic data of various avocado races [22]. In the current study, we clarified the races of uncharacterized landraces and assessed the genetic diversity among avocado landraces based on quality traits and an EST-SSR marker system.…”