The calcium-dependent protein kinase CPK1 regulates a pivotal developmental switch in senescence and leaf cell death through direct phosphorylation of transcrip

Background: Some of the major contributions to the rapid genetic improvement of commercial plant species and crops come through the identification of genetic markers linked to specific traits with a marketable value that have been difficult or impossible to identify and improve with conventional methods. Next-Generation Sequencing (NGS) is a powerful platform that has enabled the exploration of the genome in different plant species. The success of NGS is dependent on the quality of DNA or RNA extracted. Methods and commercial kits for extraction of intact nucleic acids for many model plant species are available but the need exists still for high-throughput methods suitable for most non-model plants, in particular, recalcitrant plant species. Findings: - Our group has developed a detailed method that allows the isolation of genomic DNA or total RNA from different tissues from different plant species that yield enough material suitable for high-throughput SNP genotyping, sequencing applications and traditional downstream genetic analysis. - One single operator can extract nucleic acids from 192 samples in about 3.5 hours but the protocol is completely or partially-compatible with automation depending on available equipment. - We provide tips and comments that will help to improve the quality of the isolated material and solutions for everyday laboratory problems that will save time and money. - Little variations in the protocol allow the application of the method to plant tissues from fresh, dry or frozen samples. - The protocol is easy to set up in any molecular research laboratory, uses inexpensive reagents and handmade solutions that can be stored for several months, does not require the use of liquid nitrogen, and works best with 15 - 30 mg of fresh, frozen or dry plant material. - DNA and RNA have been isolated from at least 30 species, including especially recalcitrant forest trees from angiosperm and gymnosperm families. DNA has been also extracted from stored (2 to 6 years) silica-dried samples of species of Pinaceae and Nymphaeaceae families. - The protocol is a home-made adapatation of different published protocols that can be used in combination with different nucleic acids extraction kits. For reference consult: Ivanova, N.V. et al. 2006, Ivanova, N.V. et al 2008, and Whitlock R. et al. 2008.

The Christmas tree industry is mainly supplied with seeds from natural stands and genetically improved germplasm from clonal seed orchards (CSOs). Chalcid wasps (Megastigmus spp.) have a negative effect on the value of seedlots by reducing the quantity of viable seeds and represent a risk for national and international seed trade. Seed radiographies, field observations and seed-dissections from 25 Fraser fir (Abies fraseri (Pursh) Poir.) clones were used to determine chalcid infestation in a CSO of the southern Appalachians. We provided direct evidence that Mesopolobus cf. pinus is an ectoparasitoid of M. specularis, proposed a model that linked different aspects of the biology of these species and discussed host–insect phenological relationships. Infestation of seeds in some clones across surveys, suggested a potential genetic component to chalcid wasp's prevalence. Cold and dark rearing regimens were used to identify changes in adult insect emergence after variation of overwinter conditions. Both the frequency and the time of emerging M. specularis adults from seeds were affected by the duration of the treatments but not statistical differences were found for Mesopolobus cf. pinus. We present evidence for the efficiency of a gravity-seed cleaning method eliminating upwards of 95% of chalcid-infested seeds.

For several years, studies on insect behavior, larval development, infestation patterns, parasitism, and identification of species have been possible by the implementations of methods and equipment to rear insects in controlled environments. Rearing methods vary from being simple duplication of nature, such as holding insects and their feed resources to those where years of practice are required to tuning the optimal conditions. The vast majority of the rearing protocols are derived empirically from experimenting with a variety of materials and procedures until success is achieved. Because the determination of infestation caused by Megastigmus specularis, a seed-parasite, on Fraser fir seeds and their relation to its parasitoid Mesopolobus, are fundamental to biological control and related fields, accurate data are of great importance. Our method was established to obtain chalcid alive, using an economic and fast setting-up. This method has been used in our lab for different studies related to chalcids and their parasitoids. We present a very simple method to rear these species under laboratory conditions. The protocol can be used in experiments in which variation in day length and temperature is required. The rearing mounting can be easily placed in any laboratory or inside of a more controlled environment chamber and use inexpensive materials. This rearing method facilitates the observation and capture of adult insects while maximizing the number of experimental units (EUs) that could be evaluated.