Expressed Sequence Tag Database Mining Research Articles

Homopteran Vector Biomarkers for Efficient Circulative Plant Virus Transmission are Conserved in Multiple Aphid Species and the Whitefly Bemisia tabaci

Plant viruses in the families Luteoviridae and Geminiviridae are phloem restricted and are transmitted in a persistent, circulative manner by homopteran insects. Using fluorescence 2-D difference gel electrophoresis to compare the proteomes of F2 genotypes of Schizaphis graminum segregating for virus transmission ability, we recently discovered a panel of protein biomarkers that predict vector competency. Here we used aphid and whitefly nucleotide and expressed sequence tag database mining to test whether these biomarkers are conserved in other homopteran insects. S. graminum gene homologs that shared a high degree of predicted amino acid identity were discovered in two other aphid species and in the whitefly Bemisia tabaci. Selected reaction monitoring mass spectrometry was used to validate the expression of these biomarkers proteins in multiple aphid vector species. The conservation of these proteins in multiple insect taxa that transmit plant viruses along the circulative transmission pathway creates the opportunity to use these biomarkers to rapidly identify insect populations that are the most efficient vectors and allow them to be targeted for control prior to the spread of virus within a crop.

Single nucleotide polymorphisms in several porcine cathepsin genes are associated with growth, carcass, and production traits in Italian Large White pigs1

To identify DNA markers associated with performance, carcass, and meat production traits including muscle postmortem cathepsin activity, several porcine genes encoding for lysosomal proteinases (cathepsin B, CTSB; cathepsin D, CTSD; cathepsin F, CTSF; cathepsin H, CTSH; cathepsin L, CTSL; and cathepsin Z, CTSZ) and for a cathepsin inhibitor (cystatin B) were investigated. Single nucleotide polymorphisms were identified in CTSD, CTSH, CTSL, and CTSZ genes with a combination of in silico expressed sequence tag database mining and single-strand conformation polymorphism analysis. Sequencing and PCR-RFLP protocols were used to validate the identified polymorphisms. Allele frequencies at these loci were investigated in Italian Large White, Landrace, Duroc, Piétrain, Belgian Landrace, Hampshire, and Meishan breeds. Genotyping CTSD and CTSH markers made it possible to genetically map these genes to SSC 2 and 7, respectively. Markers in CTSD, CTSH, CTSL, and CTSZ genes, together with mutations we previously reported in cystatin B, CTSB, and CTSF genes, were genotyped in an Italian Large White sib-tested population (272 or 482 animals). For these animals, meat quality traits (cathepsin B activity, pH measured at 2 h postmortem, pH measured at 24 h postmortem, glycogen, lactate, and glycolytic potential of semimembranosus muscle) and EBV for ADG, lean cuts (LC), backfat thickness (BFT), ham weight (HW), and feed:gain ratio (FGR) were determined. Analyzed markers did not show any association with muscle cathepsin B activity. Thus, it could be possible that different genes, other than these investigated candidates, affect this trait, which is correlated with the excessive softness defect of dry-cured hams. The results of association analysis confirmed the effects we already reported in another study for CTSF on ADG (P = 0.008), LC (P = 0.001), and BFT (P = 0.02). Moreover, CTSD was associated with ADG, LC (P < 0.0001), BFT, HW, and FGR (P < 0.001); CTSH was associated with FGR (P = 0.026); and CTSZ was associated with ADG (P = 0.006), LC (P = 0.01), HW (P = 0.024), and FGR (P = 0.029). The biochemical and physiological functions of the lysosomal proteinases, together with the results obtained in our investigation, suggest that the cathepsin gene family might play important roles affecting economic traits in pigs.

Characterization of 95 novel microsatellite markers for Zhikong scallop Chlamys farreri using FIASCO-colony hybridization and EST database mining

In order to construct a simple sequence repeat (SSR)-based genetic linkage map and to promote molecular marker-assisted selection (MAS) in scallop breeding, the methods of Fast Isolation by AFLP of Sequences COntaining repeats (FIASCO)-colony hybridization and expressed sequence tag (EST) database mining were modified and used to develop 95 novel microsatellite markers for Zhikong scallop. The SSR-enriched library constructed by the FIASCO method consisted of 830 clones, and 295 (35.5%) positive clones were identified after colony hybridization. One hundred and fifty clones were randomly sequenced and the results showed all clones contained at least one microsatellite. Of 91 primer pairs designed, 72 were amplified scorable polymerase chain reaction (PCR) products and 70 were polymorphic with the allele number range of 3-16 alleles/locus (average 7.0 alleles/locus). When EST database mining was performed, 66 microsatellites containing ESTs were identified from 3467 sequences deposited in GenBank. Based on cluster analysis of length and GC content of the flanking regions, 47 primer pairs were designed and 23 scorable EST SSRs were obtained. Compared with genomic SSRs developed in this study, EST SSRs showed lower genetic variability with an average of 4.2 alleles/locus. The results in the present study demonstrate that modified FIASCO-colony hybridization is an efficient and low-cost method for the isolation of large numbers of microsatellite markers for scallop species.

Development and characterization of 45 novel microsatellite markers for sea cucumber (Apostichopus japonicus)

AbstractSimple sequence repeat‐enriched library screening and expressed sequence tag database mining were adopted to develop microsatellite markers for sea cucumber (Apostichopus japonicus). Eighty‐three microsatellite loci were selected for polymorphism assessment using 48 individuals. The results showed that 45 novel loci were polymorphic. The number of alleles ranged from two to 16, and the values of observed and expected heterozygosities varied from 0 to 0.9375 and from 0.1135 to 0.9674, respectively. No significant linkage disequilibrium between pairs of loci was found and 26 loci conformed to the Hardy–Weinberg equilibrium. These markers are therefore a potential tool for studies in the population structure and linkage map construction for A. japonicus.

Identification and characterization of a Neospora caninum microneme-associated protein (NcMIC4) that exhibits unique lactose-binding properties.

Microneme proteins have been shown to play an important role in the early phase of host cell adhesion, by mediating the contact between the parasite and host cell surface receptors. In this study we have identified and characterized a lectin-like protein of Neospora caninum tachyzoites which was purified by alpha-lactose-agarose affinity chromatography. Upon separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, this lactose-binding protein migrated at 70 and 55 kDa under reducing and nonreducing conditions, respectively. Immunofluorescence and immunogold electron microscopy with affinity-purified antibodies showed that the protein was associated with the tachyzoite micronemes. Mass spectrometry analyses and expressed sequence tag database mining revealed that this protein is a member of the Neospora microneme protein family; the protein was named NcMIC4 (N. caninum microneme protein 4). Upon two-dimensional gel electrophoresis, NcMIC4 separated into seven distinct isoforms. Incubation of extracellular parasites at 37 degrees C resulted in the secretion of NcMIC4 into the medium as a soluble protein, and the secreted protein exhibited a slightly reduced M(r) but retained its lactose-binding properties. Immunofluorescence was used to investigate the temporal and spatial distribution of NcMIC4 in tachyzoites entering their host cells and showed that reexpression of NcMIC4 took place 30 min after entry into the host cell. Incubation of secreted fractions and purified NcMIC4 with Vero cells demonstrated binding of NcMIC4 to Vero cells as well as binding to chondroitin sulfate A glycosaminoglycans.

Development and Characterization of Microsatellite Markers in Sweetpotato

To promote the implementation of genetic analyses and breeding programs, a set of microsatellite markers in sweetpotato should be developed, which cover the entire sweetpotato genome. To achieve this objective, 102 polymorphic microsatellite markers were developed using three different methods: 1) screening of a small-insert genomic library, 2) construction of a microsatellite-enriched library (51-fold enrichment) and 3) mining of EST databases. Approximately 8,000 clones of the small-insert library were screened with (GA)20 and (CA)20 probes, and 42 positive clones were identified. One hundred and twenty-two clones containing microsatellites were identified from the enriched library, which consisted of 800 clones. Thirty-two and 47 primer pairs were designed for these clones containing microsatellites from the small-insert library and enriched library, respectively. Among the sweetpotato accessions examined, 27 of these microsatellite markers showed polymorphism. Eventually, 4,153 sequences from a published expressed sequence tag (EST) databases were mined as a source for the development of microsatellite markers, and 379 sequences containing microsatellites were identified. A total of 151 primer pairs were designed and 120 scorable microsatellite markers were obtained. Seventy-five EST-SSR loci showed length polymorphisms. Of these polymorphic loci, 71 % were associated with some genes.