De novo transcriptome sequencing and assembly from apomictic and sexual Eragrostis curvula genotypes.

A long-standing goal in plant breeding has been the ability to confer apomixis to agriculturally relevant species, which would require a deeper comprehension of the molecular basis of apomictic regulatory mechanisms. Eragrostis curvula (Schrad.) Nees is a perennial grass that includes both sexual and apomictic cytotypes. The availability of a reference transcriptome for this species would constitute a very important tool toward the identification of genes controlling key steps of the apomictic pathway. Here, we used Roche/454 sequencing technologies to generate reads from inflorescences of E. curvula apomictic and sexual genotypes that were de novo assembled into a reference transcriptome. Near 90% of the 49568 assembled isotigs showed sequence similarity to sequences deposited in the public databases. A gene ontology analysis categorized 27448 isotigs into at least one of the three main GO categories. We identified 11475 SSRs, and several of them were assayed in E curvula germoplasm using SSR-based primers, providing a valuable set of molecular markers that could allow direct allele selection. The differential contribution to each library of the spliced forms of several transcripts revealed the existence of several isotigs produced via alternative splicing of single genes. The reference transcriptome presented and validated in this work will be useful for the identification of a wide range of gene(s) related to agronomic traits of E. curvula, including those controlling key steps of the apomictic pathway in this species, allowing the extrapolation of the findings to other plant species

Genotypes assayed to simple sequence repeat (SSR) validation.

<p>Genotypes assayed to simple sequence repeat (SSR) validation.</p

Graphic summary of the <i>de novo-</i>assembled <i>E</i>. <i>curvula</i> transcriptome.

<p>(A) Length distribution of the assembled isotigs; (B) Number of contigs used to assemble individual isotigs; (C) Number of isotigs used to assemble individual isogroups.</p

Analysis of gene expression.

<p>Reactions were performed with cDNA from inflorescences from the genotypes Tanganyika (T) and OTA-S (O). a) RT-PCR. The primer pair used are shown over the agarose gel image. Ubiquitin conjugating enzyme transcript (UBICE) was used as housekeeping gene; molecular mass markers are shown on the left; b) qRT-PCR. The primer pairs used are shown over the figures. *—significant differences between O and T for the primer pair tested (p < 0.05).</p

Gene-based SSR markers development.

<p>Gene-based SSR markers development.</p

Classification of <i>E</i>. <i>curvula</i> annotated isotig sequences based on predicted gene ontology (GO) terms.

<p>Terms were obtained with BLAST2GO at level 2. (A) Biological process; (B) molecular function; (C) cellular component.</p

Description of the raw and filtered reads for the four obtained libraries.

<p>Description of the raw and filtered reads for the four obtained libraries.</p

Schematic representation of an example of alternative splicing identified for the isogruop01298.

<p>Image shows the alignment among a genomic sequence from <i>Zea maize</i> (AECO01010067.1) and isotig13648, isotig13649 and seven mRNA sequences from <i>Z</i>. <i>maize</i> (NM_001159013), <i>Brachypodium distachyon</i> (XM_003563288.3), <i>Sorghum bicolor</i> (XM_002437369.1), <i>Hordeum vulgare</i> (AK365976.1), <i>Oryza sativa</i> (XM_015787382.1), <i>Setaria italica</i> (XM_004965571.3), <i>Triticum aestivum</i> (AK335082.1) proving evidence of possible alternative splicing in the <i>E</i>. <i>curvula</i> sequences under study. Numbers indicates the position in the queried sequence.</p

Classification of simple sequence repeats (SSRs) according to repeat unit size.

<p>The graph illustrates the frequency of different sized SSR units classified as mononucleotides (Mono-), dinucleotides (Di-), trinucleotides (Tri-), tetranucleotides (Tetra-), pentanucleotides (Penta-) and hexanucleotides (Hexa-) across the assembled <i>E</i>. <i>curvula</i> reference transcriptome.</p

A representative profile obtained with simple sequence repeat (SSR) markers designed based on the assembled isotigs in different <i>E</i>. <i>curvula</i> cultivars.

<p>Acrylamide gel showing amplification products obtained in the 14 assayed genotypes using the primers designed based on: a) isotig06745; b) isotig28255; c) isotig 35118. References: M (Marker), B (Negative).</p