Microsatellite markers have played a major role in ecological, evolutionary and conservation research during the past 20 years. However, technical constrains related to the use of capillary electrophoresis and a recent technological revolution that has impacted other marker types have brought to question the continued use of microsatellites for certain applications. We present a study for improving microsatellite genotyping in ecology using high-throughput sequencing (HTS). This approach entails selection of short markers suitable for HTS, sequencing PCR-amplified microsatellites on an Illumina platform and bioinformatic treatment of the sequence data to obtain multilocus genotypes. It takes advantage of the fact that HTS gives direct access to microsatellite sequences, allowing unambiguous allele identification and enabling automation of the genotyping process through bioinformatics. In addition, the massive parallel sequencing abilities expand the information content of single experimental runs far beyond capillary electrophoresis. We illustrated the method by genotyping brown bear samples amplified with a multiplex PCR of 13 new microsatellite markers and a sex marker. HTS of microsatellites provided accurate individual identification and parentage assignment and resulted in a significant improvement of genotyping success (84%) of faecal degraded DNA and costs reduction compared to capillary electrophoresis. The HTS approach holds vast potential for improving success, accuracy, efficiency and standardization of microsatellite genotyping in ecological and conservation applications, especially those that rely on profiling of low-quantity/quality DNA and on the construction of genetic databases. We discuss and give perspectives for the implementation of the method in the light of the challenges encountered in wildlife studies.
Iron is an essential element for plant metabolism because of its redox properties. Its long distance and intracellular trafficking require specialized proteins and low molecular mass chelates because of its insolubility and toxicity in presence of oxygen. Iron deficiency induces various morphological and biochemical changes. They include root hair morphogenesis, differentiation of rhizoder-ma1 cells into transfer cells, yellowing of leaves and ultrastructural disorganisation of chloroplasts and mitochondria, as well as increased synthesis of organic acids and phenolics, and activation of root systems responsible for an enhanced iron uptake capacity. Upon iron resupply, these alterations disappeared within few days and a transient accumulation of the iron storage protein ferritin in the plastids is one of the early events in this process. Iron excess can also occur in plants where it elicits an oxidative stress leading to necrotic spots in the leaves. Induction of ferritin synthesis is again an early event of the plant response to this iron toxicity. Plant hormones such as auxin, abscisic acid and ethylene, as well as reactive oxygen intermediates play an important role in the transduction pathways, allowing plants to respond to these iron-deficiency and excess stresses. Similarities and differences among the various mechanisms responsible for iron uptake and storage in mammals, higher plants and yeast are outlined. Relationships between iron and copper metabolism are also indicated.
plant / root / chloroplast / iron / ferritinIron traffk in non-stressed plants
Iron concentration and ferritin distribution have been determined in different organs of pea (Pisum sativum) during development under conditions of continuous iron supply from hydroponic cultures. No ferritin was detected in total protein extracts from roots or leaves. However, a transient iron accumulation in the roots, which corresponds to an increase in iron uptake, was observed when young fruits started to develop. Ferritin was detectable in total protein extracts of flowers and pods, and it accumulated in seeds. In seeds, the same relative amount of ferritin was detected in cotyledons and in the embryo axis. In cotyledons, ferritin and iron concentration decrease progressively during the first week of germination. Ferritin in the embryo axis was processed, and disappeared, during germination, within the first 4 days of radicle and epicotyl growth. This degradation of ferritin in vivo was marked by a shortening of a 28 kDa subunit, giving 26.5 and 25 kDa polypeptides, reminiscent of the radical damage occurring in pea seed ferritin during iron exchange in vitro [Laulhere, Laboure & Briat (1989) J. Biol. Chem. 264, 3629-3635]. Developmental control of iron concentration and ferritin distribution in different organs of pea is discussed.
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