Isolation and characterization of novel microsatellite markers and their application for diversity assessment in cultivated groundnut (Arachis hypogaea)

Background: Cultivated peanut or groundnut (Arachis hypogaea L.) is the fourth most important oilseed crop in the world, grown mainly in tropical, subtropical and warm temperate climates. Due to its origin through a single and recent polyploidization event, followed by successive selection during breeding efforts, cultivated groundnut has a limited genetic background. In such species, microsatellite or simple sequence repeat (SSR) markers are very informative and useful for breeding applications. The low level of polymorphism in cultivated germplasm, however, warrants a need of larger number of polymorphic microsatellite markers for cultivated groundnut. Results: A microsatellite- enriched library was constructed from the genotype TMV2. Sequencing of 720 putative SSR-positive clones from a total of 3,072 provided 490 SSRs. 71.2% of these SSRs were perfect type, 13.1% were imperfect and 15.7% were compound. Among these SSRs, the GT/CA repeat motifs were the most common (37.6%) followed by GA/CT repeat motifs (25.9%). The primer pairs could be designed for a total of 170 SSRs and were optimized initially on two genotypes. 104 (61.2%) primer pairs yielded scorable amplicon and 46 (44.2%) primers showed polymorphism among 32 cultivated groundnut genotypes. The polymorphic SSR markers detected 2 to 5 alleles with an average of 2.44 per locus. The polymorphic information content (PIC) value for these markers varied from 0.12 to 0.75 with an average of 0.46. Based on 112 alleles obtained by 46 markers, a phenogram was constructed to understand the relationships among the 32 genotypes. Majority of the genotypes representing subspecies hypogaea were grouped together in one cluster, while the genotypes belonging to subspecies fastigiata were grouped mainly under two clusters. Conclusion. Newly developed set of 104 markers extends the repertoire of SSR markers for cultivated groundnut. These markers showed a good level of PIC value in cultivated germplasm and therefore would be very useful for germplasm analysis, linkage mapping, diversity studies and phylogenetic relationships in cultivated groundnut as well as related Arachis species.

Completion of the genome sequence of Lettuce necrotic yellows virus, type species of the genus Cytorhabdovirus

We completed the genome sequence of Lettuce necrotic yellows virus (LNYV) by determining the nucleotide sequences of the 4a (putative phosphoprotein), 4b, M (matrix protein), G (glycoprotein) and L (polymerase) genes. The genome consists of 12,807 nucleotides and encodes six genes in the order 3′ leader-N-4a(P)-4b-M-G-L-5′ trailer. Sequences were derived from clones of a cDNA library from LNYV genomic RNA and from fragments amplified using reverse transcription-polymerase chain reaction. The 4a protein has a low isoelectric point characteristic for rhabdovirus phosphoproteins. The 4b protein has significant sequence similarities with the movement proteins of capillo- and trichoviruses and may be involved in cell-to-cell movement. The putative G protein sequence contains a predicted 25 amino acids signal peptide and endopeptidase cleavage site, three predicted glycosylation sites and a putative transmembrane domain. The deduced L protein sequence shows similarities with the L proteins of other plant rhabdoviruses and contains polymerase module motifs characteristic for RNA-dependent RNA polymerases of negative-strand RNA viruses. Phylogenetic analysis of this motif among rhabdoviruses placed LNYV in a group with other sequenced cytorhabdoviruses, most closely related to Strawberry crinkle virus.

Characterization of disease resistance gene candidates of the nucleotide binding site (NBS) type from banana and correlation of a transcriptional polymorphism with resistance to Fusarium oxysporum f.sp. cubense race 4

Most plant disease resistance (R) genes encode proteins with a nucleotide binding site and leucine-rich repeat structure (NBS-LRR). In this study, degenerate primers were used to amplify genomic NBS-type sequences from wild banana (Musa acuminata ssp. malaccensis) plants resistant to the fungal pathogen Fusarium oxysporum formae specialis (f. sp.) cubense (FOC) race 4. Five different classes of NBS-type sequences were identified and designated as resistance gene candidates (RGCs). The deduced amino acid sequences of the RGCs revealed the presence of motifs characteristic of the majority of known plant NBS-LRR resistance genes. Structural and phylogenetic analyses grouped the banana RGCs within the non-TIR (homology to Toll/interleukin-1 receptors) subclass of NBS sequences. Southern hybridization showed that each banana RGC is present in low copy number. The expression of the RGCs was assessed by RT-PCR in leaf and root tissues of plants resistant or susceptible to FOC race 4. RGC1, 3 and 5 showed a constitutive expression profile in both resistant and susceptible plants whereas no expression was detected for RGC4. Interestingly, RGC2 expression was found to be associated only to FOC race 4 resistant lines. This finding could assist in the identification of a FOC race 4 resistance gene.

High global diversity of cycloviruses amongst dragonflies

Members of the family Circoviridae, specifically the genus Circovirus, were thought to infect only vertebrates; however, members of a sister group under the same family, the proposed genusCyclovirus, have been detected recently in insects. In an effort to explore the diversity of cycloviruses and better understand the evolution of these novel ssDNA viruses, here we present five cycloviruses isolated from three dragonfly species (Orthetrum sabina, Xanthocnemis zealandica and Rhionaeschna multicolor) collected in Australia, New Zealand and the USA, respectively. The genomes of these five viruses share similar genome structure to other cycloviruses, with a circular ~1.7 kb genome and two major bidirectionally transcribed ORFs. The genomic sequence data gathered during this study were combined with all cyclovirus genomes available in public databases to identify conserved motifs and regulatory elements in the intergenic regions, as well as determine diversity and recombinant regions within their genomes. The genomes reported here represent four different cyclovirus species, three of which are novel. Our results confirm that cycloviruses circulate widely in winged-insect populations; in eight different cyclovirus species identified in dragonflies to date, some of these exhibit a broad geographical distribution. Recombination analysis revealed both intra-and inter-species recombination events amongst cycloviruses, including genomes recovered from disparate sources (e.g. goat meat and human faeces). Similar to other well-characterized circular ssDNA viruses, recombination may play an important role in cyclovirus evolution. © 2013 SGM.

Resistance gene analogues in sugarcane and sorghum and their association with quantitative trait loci for rust resistance

Fifty-four different sugarcane resistance gene analogue (RGA) sequences were isolated, characterized, and used to identify molecular markers linked to major disease-resistance loci in sugarcane. Ten RGAs were identified from a sugarcane stem expressed sequence tag (EST) library; the remaining 44 were isolated from sugarcane stem, leaf, and root tissue using primers designed to conserved RGA motifs. The map location of 31 of the RGAs was determined in sugarcane and compared with the location of quantitative trait loci (QTL) for brown rust resistance. After 2 years of phenotyping, 3 RGAs were shown to generate markers that were significantly associated with resistance to this disease. To assist in the understanding of the complex genetic structure of sugarcane, 17 of the 31 RGAs were also mapped in sorghum. Comparative mapping between sugarcane and sorghum revealed syntenic localization of several RGA clusters. The 3 brown rust associated RGAs were shown to map to the same linkage group (LG) in sorghum with 2 mapping to one region and the third to a region previously shown to contain a major rust-resistance QTL in sorghum. These results illustrate the value of using RGAs for the identification of markers linked to disease resistance loci and the value of simultaneous mapping in sugarcane and sorghum.