Analysis of cDNA transcripts from Coniothyrium minitans reveals a diverse array of genes involved in key processes during sclerotial mycoparasitism

Coniothyrium minitans colonises and destroys the sclerotia of Sclerotinia sclerotiorum in nature exhibiting ecologically obligate mycoparasitism as its spores remain dormant in soil and only grow actively in the presence of the sclerotia. Molecular mechanisms underlying sclerotial mycoparasitism are poorly defined. We identified 251 unisequences representing genes preferentially expressed by C. minitans during sclerotial mycoparasitism, substantially increasing the molecular knowledge of this commercially important biocontrol agent. Genes associated with signalling and cellular communication, degradation of host cell walls and energy reserves, nutrient utilisation, detoxification and stress response were identified suggesting that C. minitans employs a number of key processes during host colonisation. Several of these genes are novel to fungal-fungal interactions (e.g. PTH11-like GPCR and the ETP gene cluster). Secretin receptor-like GPCR and the TGF-beta signalling system have not yet been characterised in filamentous fungi. This study provides the basis for in-depth gene function analysis in sclerotial mycoparasitism.     

Expression and genomic imprinting of the porcine Rasgrf1 gene

Imprinted genes play important roles in mammalian growth, development and behavior. The Rasgrf1 (Ras protein-specific guanine nucleotide exchange factor 1) gene has been identified as an imprinted gene in mouse and rat. In the present study, we detected its sequence, imprinting status and expression pattern in the domestic pigs. A 228 bp partial sequence located in exon 14 and a 193 bp partial sequence located in exon 1 of the Rasgrf1 gene in domestic pigs were obtained. A G/A transition, was identified in Rasgrf1 exon 14, and then, the reciprocal Berkshire × Wannan black F₁ hybrid model and the RT-PCR-RFLP method were used to detect the imprinting status of porcine Rasgrf1 gene at the developmental stage of 1-day-old. The expression profile results indicated that the porcine Rasgrf1 mRNA was highly expressed in brain, pituitary and pancreas, followed by kidney, stomach, lung, testis, small intestine, ovary, spleen and liver, and at low levels of expression in longissimus dorsi, heart, and backfat. The expression levels of Rasgrf1 gene in brain, pituitary and pancreas tissues were significantly different between the two reciprocal F₁ hybrids. Imprinting analysis showed that porcine Rasgrf1 gene was maternally expressed in the liver, small intestine, paternally expressed in the lung, but biallelically expressed in brain, heart, spleen, kidney, stomach, pancreas, backfat, testis, ovary, longissimus dorsi and pituitary tissues.     

A high-throughput apple SNP genotyping platform using the GoldenGate™ assay

EST data generated from 14 apple genotypes were downloaded from NCBI and mapped against a reference EST assembly to identify Single Nucleotide Polymorphisms (SNPs). Mapping of these SNPs was undertaken using 90% of sequence similarity and minimum coverage of four reads at each SNP position. In total, 37,807 SNPs were identified with an average of one SNP every 187 bp from a total of 6888 unique EST contigs. Identified SNPs were checked for flanking sequences of ≥ 60 bp along both sides of SNP alleles for reliable design of a custom high-throughput genotyping assay. A total of 12,299 SNPs, representing 6525 contigs, fit the selected criterion of ≥ 60 bp sequences flanking a SNP position. Of these, 1411 SNPs were validated using four apple genotypes. Based on genotyping assays, it was estimated that 60% of SNPs were valid SNPs, while 26% of SNPs might be derived from paralogous regions.