De novo assembly and characterization of leaf transcriptome for the development of functional molecular markers of the extremophile multipurpose tree species Prosopis alba

Abstract

BackgroundProsopis alba (Fabaceae) is an important native tree adapted to arid and semiarid regions of north-western Argentina which is of great value as multipurpose species. Despite its importance, the genomic resources currently available for the entire Prosopis genus are still limited. Here we describe the development of a leaf transcriptome and the identification of new molecular markers that could support functional genetic studies in natural and domesticated populations of this genus.ResultsNext generation DNA pyrosequencing technology applied to P. alba transcripts produced a total of 1,103,231 raw reads with an average length of 421 bp. De novo assembling generated a set of 15,814 isotigs and 71,101 non-assembled sequences (singletons) with an average of 991 bp and 288 bp respectively. A total of 39,000 unique singletons were identified after clustering natural and artificial duplicates from pyrosequencing reads.Regarding the non-redundant sequences or unigenes, 22,095 out of 54,814 were successfully annotated with Gene Ontology terms. Moreover, simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs) were searched, resulting in 5,992 and 6,236 markers, respectively, throughout the genome. For the validation of the the predicted SSR markers, a subset of 87 SSRs selected through functional annotation evidence was successfully amplified from six DNA samples of seedlings. From this analysis, 11 of these 87 SSRs were identified as polymorphic. Additionally, another set of 123 nuclear polymorphic SSRs were determined in silico, of which 50% have the probability of being effectively polymorphic.ConclusionsThis study generated a successful global analysis of the P. alba leaf transcriptome after bioinformatic and wet laboratory validations of RNA-Seq data.The limited set of molecular markers currently available will be significantly increased with the thousands of new markers that were identified in this study. This information will strongly contribute to genomics resources for P. alba functional analysis and genetics. Finally, it will also potentially contribute to the development of population-based genome studies in the genera.

De Novo Transcriptome Analysis of Dalbergia odorifera T. Chen (Fabaceae) and Transferability of SSR Markers Developed from the Transcriptome

“…This may be because they all belong to the family Fabaceae and share a high level of genomes sequence similarity. Similar observations have been reported on other transcriptome studies [41,42]. It is thought that KEGG provides a basic platform for systematic analysis of gene function in terms of gene product networks [43].…”

“…Therefore, the development of a known polymorphic SSR marker database for D. odorifera is of great use for diversity studies and breeding programs, as well as being transferable to closely Dalbergia species. In the present study, we identified 35,774 putative SSRs from the transcriptome dataset, which is of great differences in numbers, dominant repeats, and motif types reported from other legume species using Illumina sequencing [41,42,44]. This may be attributable to inconsistencies in genome structure or composition, dataset size, search method, or criteria.…”

Genetic diversity and structure of the vulnerable species Prosopis chilensis (Molina) Stuntz in the Coquimbo Region, Chile

“…This chloroplast marker presented a specific allele pattern for the Choapa valley that did not reveal population structure, which is consistent with the weak structure detected by the nuclear SSR markers. To our knowledge, the eight chloroplast SSR markers tested in this work had not been used previously to characterize P. chilensis populations, although other chloroplast SSR markers from P. alba had been previously described (Torales et al 2013). As chloroplast DNA is mainly maternally inherited, information from chloroplast microsatellites differs from that obtained from nuclear SSR because cytoplasmic genomes are inherited uniparentally in most angiosperms (Birky 1995).…”

“…The quality of the obtained DNA was evaluated on 0.8% agarose gels stained with ethidium bromide and DNA concentration was measured with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Inc. Wilmington, DE, USA), according to the user's manual. (Mottura et al 2005, Bessega et al 2013, Torales et al 2013, Alves et al 2014, Pomponio et al 2015. This study was based on six nuclear microsatellite loci: Mo05, Mo07, Mo08, Mo09, Mo13, and Mo16, described for P. chilensis by Mottura et al (2005).…”

De novo assembly, functional annotation, and marker development of Asian pear (Pyrus pyrifolia) fruit transcriptome through massively parallel sequencing

“…Next-generation sequencing technologies have increased the opportunity for molecular marker development in non-model study organisms on an unprecedented scale (Torales et al, 2013). To date, only a few genetic maps have been developed for Pyrus spp (Montanari et al, 2013).…”