Assessment of SNPs for linkage mapping in Eucalyptus: construction of a consensus SNP/microsatellite map from two unrelated pedigrees

Financial support. Brazilian Ministry of Science and Technology (CNPq Grant 577047-2008-6), FAP-DF NEXTREE Grant 193.000.570/2009 and EMBRAPA Macroprogram 2 project grant 02.07.01.004

Transcriptome Analysis in Cotton Boll Weevil (<i>Anthonomus grandis</i>) and RNA Interference in Insect Pests

<div><p>Cotton plants are subjected to the attack of several insect pests. In Brazil, the cotton boll weevil, <i>Anthonomus grandis</i>, is the most important cotton pest. The use of insecticidal proteins and gene silencing by interference RNA (RNAi) as techniques for insect control are promising strategies, which has been applied in the last few years. For this insect, there are not much available molecular information on databases. Using 454-pyrosequencing methodology, the transcriptome of all developmental stages of the insect pest, <i>A. grandis</i>, was analyzed. The <i>A. grandis</i> transcriptome analysis resulted in more than 500.000 reads and a data set of high quality 20,841 contigs. After sequence assembly and annotation, around 10,600 contigs had at least one BLAST hit against NCBI non-redundant protein database and 65.7% was similar to <i>Tribolium castaneum</i> sequences. A comparison of <i>A. grandis</i>, <i>Drosophila melanogaster</i> and <i>Bombyx mori</i> protein families’ data showed higher similarity to dipteran than to lepidopteran sequences. Several contigs of genes encoding proteins involved in RNAi mechanism were found. PAZ Domains sequences extracted from the transcriptome showed high similarity and conservation for the most important functional and structural motifs when compared to PAZ Domains from 5 species. Two SID-like contigs were phylogenetically analyzed and grouped with <i>T. castaneum</i> SID-like proteins. No RdRP gene was found. A contig matching chitin synthase 1 was mined from the transcriptome. dsRNA microinjection of a chitin synthase gene to <i>A. grandis</i> female adults resulted in normal oviposition of unviable eggs and malformed alive larvae that were unable to develop in artificial diet. This is the first study that characterizes the transcriptome of the coleopteran, <i>A. grandis</i>. A new and representative transcriptome database for this insect pest is now available. All data support the state of the art of RNAi mechanism in insects.</p> </div

Genes involved in RNAi mechanism found in <i>A. grandis</i> transcriptome.

<p>The comparison with genes of <i>C. elegans</i>, <i>T. castaneum</i>, and <i>D. melanogaster</i> suggested that RNAi mechanism is well conserved in insects (A, B, C, D), including lack of amplification (E). No gene involved in dsRNA degradation was found (F). The number of contigs found in <i>A. grandis</i> transcriptome for each gene class is shown. </p

Distance neighbor-joining tree showing the phylogeny of a SID-like contig of <i>A. grandis</i> (A_grandis_454_c2889) and SID-like proteins of the insects <i>T. castaneum</i>, <i>B. impatiens</i>, <i>A. mellifera</i>, <i>L. migratoria</i>, <i>B. mori</i>, <i>A. gossypii</i>, <i>H. saltator</i>, <i>Camponotus floridanus</i>.

<p>The percentage of percentage of bootstrap confidence values is shown at the nodes. </p

Comparison of dicer and argonaute PAZ domains.

<p>Two cotton boll weevil contigs were aligned to five species sequences: <i>D. melanogaster</i> (Dm_Dicer-1, Dm_AGO1C, Dm_AGO2)<i>, C. elegans</i> (Ce_Dicer1, Ce_Alg1, Ce_Alg2)<i>, Homo sapiens</i> (Hs_Dicer-1, Hs_Ago1)<i>, A. thaliana</i> (At_Dicer-like-1, At_AGO, At_AGO1) and <i>Schizosaccharomyces pombe</i> (Sp_AGO1). The sequence IDs are the same found in the NCBI Protein Database. Secondary structures within the domain are indicated as α-helices and β structures. The highlighted residues are responsible for the stabilization of the dsRNA-binding region. In yellow, a subdomain of aromatic residues. Along with a cysteine residue (blue), preceded by a proline and a glutamate (yellow), some invariant residues (red) create a hydrophobic subdomain that interacts with RNA. Residues that differ in dicer and argonaute PAZ domains are shown in brown. </p

Effect of AntgCHS1 on <i>A. grandis</i> on oviposition.

<p>Larvae that emerged from eggs laid by females previously microinjected with 200 ng of either GUS (control) or AntgCHS1 dsRNA (A). After egg hatching, larvae were fed in artificial diet for 7 days. Details of head capsule show malformations in AntgCHS1 dsRNA-treated larvae (C and D) when compared to control (B). The viability was reduced (E) and as well as the number of transcripts of AntgCHS1 (F) in eggs laid by females previously microinjected with AntgCHS1 dsRNA.</p

Comparison of the distribution of GO terms.

<p>The X-axis shows subgroups of cellular component, molecular functions and biological process from GO. Distribution of GO terms of gene families of <i>T. castaneum</i> and <i>A. grandis</i> are compared. The Y-axis shows the percentage (left) and the number of genes (right) of the matched Pfam entries. </p