Transformation of pea (Pisum sativum L.) byAgrobacterium tumefaciens

Explants fromPisum sativum shoot cultures and epicotyls were transformed by cocultivation withAgrobacterium tumefaciens vectors carrying plant selectable markers and transformants could be selected on a medium containing kanamycin. Transformants could also be obtained at a low frequency by cocultivating small protoplast-derived colonies. The transformed nature of the calli obtained from selection was confirmed by opine assay and DNA analysis. In addition five cultivars of pea were tested for their response to seven differentAgrobacterium tumefaciens strains. The response pattern coincided largely between the different pea cultivars, being more dependent on the bacterial strain than the cultivar used.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-benzyladenine - Km kanamycin - NAA -naphthaleneacetic acid - NOS nopaline synthase - NPT neomycin phosphotransferase - OCS octopine synthase     

Purification and biochemical characterization of an extracellular serine peptidase from Aspergillus terreus

Peptidases are important because they play a central role in pharmaceutical, food, environmental, and other industrial processes. A serine peptidase from Aspergillus terreus was isolated after two chromatography steps that showed a yield of 15.5%. Its molecular mass was determined to be 43 kD, by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). This peptidase was active between pH 5.0 to 8.0 and had maximum activity at pH 7.0, at 45°C. When exposited with 1 M of urea, the enzyme maintained 100% activity and used azocasein as substrate. The N-terminal (first 15 residues) showed 33% identity with the serine peptidase of Aspergillus clavatus ES1. The kinetics assays showed that subsite S₂ did not bind polar basic amino acids (His and Arg) nonpolar acidic amino acids (Asp and Glu). The subsite S₁ showed higher catalytic efficiency than the S₂ and S₃ subsites.     

Estimating Additive and Non-Additive Genetic Variances and Predicting Genetic Merits Using Genome-Wide Dense Single Nucleotide Polymorphism Markers

Non-additive genetic variation is usually ignored when genome-wide markers are used to study the genetic architecture and genomic prediction of complex traits in human, wild life, model organisms or farm animals. However, non-additive genetic effects may have an important contribution to total genetic variation of complex traits. This study presented a genomic BLUP model including additive and non-additive genetic effects, in which additive and non-additive genetic relation matrices were constructed from information of genome-wide dense single nucleotide polymorphism (SNP) markers. In addition, this study for the first time proposed a method to construct dominance relationship matrix using SNP markers and demonstrated it in detail. The proposed model was implemented to investigate the amounts of additive genetic, dominance and epistatic variations, and assessed the accuracy and unbiasedness of genomic predictions for daily gain in pigs. In the analysis of daily gain, four linear models were used: 1) a simple additive genetic model (MA), 2) a model including both additive and additive by additive epistatic genetic effects (MAE), 3) a model including both additive and dominance genetic effects (MAD), and 4) a full model including all three genetic components (MAED). Estimates of narrow-sense heritability were 0.397, 0.373, 0.379 and 0.357 for models MA, MAE, MAD and MAED, respectively. Estimated dominance variance and additive by additive epistatic variance accounted for 5.6% and 9.5% of the total phenotypic variance, respectively. Based on model MAED, the estimate of broad-sense heritability was 0.506. Reliabilities of genomic predicted breeding values for the animals without performance records were 28.5%, 28.8%, 29.2% and 29.5% for models MA, MAE, MAD and MAED, respectively. In addition, models including non-additive genetic effects improved unbiasedness of genomic predictions.     

Genome-wide and local pattern of linkage disequilibrium and persistence of phase for 3 Danish pig breeds

Background

A genome wide association study for litter size in Norwegian White Sheep (NWS) was conducted using the recently developed ovine 50K SNP chip from Illumina. After genotyping 378 progeny tested artificial insemination (AI) rams, a GWAS analysis was performed on estimated breeding values (EBVs) for litter size.

Results

A QTL-region was identified on sheep chromosome 5, close to the growth differentiation factor 9 (GDF9), which is known to be a strong candidate gene for increased ovulation rate/litter size. Sequencing of the GDF9 coding region in the most extreme sires (high and low BLUP values) revealed a single nucleotide polymorphism (c.1111G>A), responsible for a Val→Met substitution at position 371 (V371M). This polymorphism has previously been identified in Belclare and Cambridge sheep, but was not found to be associated with fertility. In our NWS-population the c.1111G>A SNP showed stronger association with litter size than any other single SNP on the Illumina 50K ovine SNP chip. Based on the estimated breeding values, daughters of AI rams homozygous for c.1111A will produce minimum 0.46 - 0.57 additional lambs compared to daughters of wild-type rams.

Conclusion

We have identified a missense mutation in the bioactive part of the GDF9 protein that shows strong association with litter size in NWS. Based on the NWS breeding history and the marked increase in the c.1111A allele frequency in the AI ram population since 1983, we hypothesize that c.1111A allele originate from Finnish landrace imported to Norway around 1970. Because of the widespread use of Finnish landrace and the fact that the ewes homozygous for the c.1111A allele are reported to be fertile, we expect the commercial impact of this mutation to be high.     

SET domain proteins in plant development

Post-translational methylation of lysine residues on histone tails is an epigenetic modification crucial for regulation of chromatin structure and gene expression in eukaryotes. The majority of the histone lysine methyltransferases (HKMTases) conferring such modifications are proteins with a conserved SET domain responsible for the enzymatic activity. The SET domain proteins in the model plant Arabidopsis thaliana can be assigned to evolutionarily conserved classes with different specificities allowing for different outcomes on chromatin structure. Here we review the present knowledge of the biochemical and biological functions of plant SET domain proteins in developmental processes. This article is part of a Special Issue entitled: Epigenetic control of cellular and developmental processes in plants.     

Regeneration of Panax ginseng C.A. Meyer by organogenesis and nuclear DNA analysis of regenerants

Plant regeneration ability of ginseng (t Panax ginseng C.A. Meyer) via organogenesis was studied. Compact callus was induced from four different types of explants-leaf, petiole, flower stalk, and root of t in vitro-grown plantlets. Petioles were found to be the best material for callus induction. Calli induced on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (4.5 μM) and kinetin (0.46 μM) were conditioned for two weeks on the same medium. These calli differentiated into adventitious shoots when cultured on 1/2MS basal medium plus kinetin 4.7 μM and silver thiosulphate 10 μM. An addition of GA₃ (2.9 μM) and BA (4.4 μM) to MS basal medium, however, induced high frequency t in vitro flowering (86.1%) and multiple shoot budding which affected the normal complete development of plantlets. Plantlets with a well-developed root system were obtained six weeks after regenerated shoots had been transplanted to 1/2 MS20 medium containing IBA 1.2 μM. Nuclear DNA content was measured to check the stability of their ploidy level. Based on DNA flow cytometric analysis, all the regenerants were typically diploids as the mother plants were, indicating that nuclear DNA content remained stable during cell differentiation. This revised version was published online in June 2006 with corrections to the Cover Date.