Semi-continuous monitoring of HMWPAH in microalgae cultures by PT-SPE/HPLC/FD-UV: Estimation of the degradation constant.

A degradation study has been performed with Selenastrum capricornutum incubated with benzo[a]anthracene and benzo[a]pyrene at 50, 100 and 266 ng mL-1 in liquid cultures. After incubation, these high molecular weight polycyclic aromatic hydrocarbons (HMW PAH) were extracted from both, the medium and biomass in a single step, and then quantified by a sensitive and validated analytical methodology based on pipette-tip SPE and HPLC with fluorescence and UV detection (PT-SPE/HPLC/FD-UV). The methodology presented good linearity r2 > 0.99, LOD of 0.9 and 0.7 ng mL-1 for BaA and BaP, respectively. A fast and semi-continuous appreciation of the degradation behavior was achieved. The pollutants were monitored at different times (0.5-18 h) in the same culture flask, with sampling volume of 1 mL. Biodegradation percentages close to-90% were observed at 18 h. The degradation curves were fitted to the first order reaction (r2 > 0.95) and the degradation rate constants were similar in all bioassays (0.1 h-1) and independent of concentration and compound. The degradation pathways of HMW PAH by microalgae and their enzyme are poorly known but the hypothesis of the degrading enzyme proportionally activated according to the PAH concentration is supported by this result. The early emergence dihydrodiol-type metabolites were detected.

Molecular and genetic characterization of the Ryadg locus on chromosome XI from Andigena potatoes conferring extreme resistance to potato virus Y.

KEY MESSAGE: We have elucidated the Andigena origin of the potato Ryadg gene on chromosome XI of CIP breeding lines and developed two marker assays to facilitate its introgression in potato by marker-assisted selection. Potato virus Y (PVY) is causing yield and quality losses forcing farmers to renew periodically their seeds from clean stocks. Two loci for extreme resistance to PVY, one on chromosome XI and the other on XII, have been identified and used in breeding. The latter corresponds to a well-known source of resistance (Solanum stoloniferum), whereas the one on chromosome XI was reported from S. stoloniferum and S. tuberosum group Andigena as well. To elucidate its taxonomic origin in our breeding lines, we analyzed the nucleotide sequences of tightly linked markers (M45, M6) and screened 251 landraces of S. tuberosum group Andigena for the presence of this gene. Our results indicate that the PVY resistance allele on chromosome XI in our breeding lines originated from S. tuberosum group Andigena. We have developed two marker assays to accelerate the introgression of Ryadg gene into breeding lines by marker-assisted selection (MAS). First, we have multiplexed RYSC3, M6 and M45 DNA markers flanking the Ryadg gene and validated it on potato varieties with known presence/absence of the Ryadg gene and a progeny of 6,521 individuals. Secondly, we developed an allele-dosage assay particularly useful to identify multiplex Ryadg progenitors. The assay based on high-resolution melting analysis at the M6 marker confirmed Ryadg plex level as nulliplex, simplex and duplex progenitors and few triplex progenies. These marker assays have been validated and can be used to facilitate MAS in potato breeding.

Robust and inexpensive SSR markers analyses using LI-COR DNA analyzer.

Plant genotyping is performed for different purposes which dictate to a large extent the type of molecular makers and platform to be used. The level of throughput, the technical capacity of the genotyping facility, and the availability of reagents are also part of the decision towards a particular genotyping system. SSR markers are quite popular markers because they are easily implementable in standard laboratories, can be used on manual gel electrophoresis, require inexpensive reagents, are mostly randomly distributed in the genome, can be located within genes, have a good discriminatory power, and are codominant with Mendelian inheritance. These features have made SSR the marker of choice for low-resolution genetic mapping and genetic diversity studies including genetic identity verification. The LI-COR platform offers both qualitative and quantitative improvements over the conventional assays based on agarose and polyacrylamide (PAGE) gels with DNA stained with ethidium bromide and silver or radiolabeled. A fast run coupled with an automated detection system using fluorophores makes possible to achieve routinely in our genotyping facility five runs per day using the same gel up to four times which results in 48 genotypes genotyped with ten SSR markers (two per gel electrophoresis using low-cost M13-tailed primers). This gel-base, low cost per sample and equipment, and medium throughput makes the LI-COR platform -particularly useful for laboratories with intermediate skills and expectations in molecular genetics.

The single Andigenum origin of Neo-Tuberosum potato materials is not supported by microsatellite and plastid marker analyses.

Neo-Tuberosum refers to cultivated potato adapted to long-day tuberization and a syndrome of related morphological and physiological traits, developed by intercrossing and selection of short-day adapted potatoes of the Solanum tuberosum Andigenum Group, native from the Andes of western Venezuela to northern Argentina. This re-creation of the modern potato helped support the theory of an Andigenum Group origin of potato in temperate regions and the possibility to access the largely untapped diversity of the Andigenum Group germplasm by base broadening breeding. This Neo-Tuberosum derived theory, the re-creation of the modern potato from Andigenum germplasm, has been universally accepted for almost 40 years, and has had tremendous impact in planning some breeding programs and supporting phylogenetic conclusions in cultivated potato. We show, with microsatellite (simple sequence repeat, SSR) and plastid DNA marker data, that Neo-Tuberosum germplasm is closely related to Chilotanum Group landraces from lowland south-central Chile rather than to Andigenum Group germplasm. We interpret this quite unexpected result to be caused by strong rapid selection against the original Andigenum clones after unintended hybridization with Chilotanum Group germplasm. In addition, we show that Neo-Tuberosum and Andigenum Group germplasm did not serve to broaden the overall genetic diversity of advanced potato varieties, but rather that Neo-Tuberosum lines and lines not using this germplasm are statistically identical with regard to genetic diversity as assessed by SSRs. These results question the long-standing Neo-Tuberosum derived theory and have implications in breeding programs and phylogenetic reconstructions of potato.

Extensive simple sequence repeat genotyping of potato landraces supports a major reevaluation of their gene pool structure and classification.

Contrasting taxonomic treatments of potato landraces have continued over the last century, with the recognition of anywhere from 1 to 21 distinct Linnean species, or of Cultivar Groups within the single species Solanum tuberosum. We provide one of the largest molecular marker studies of any crop landraces to date, to include an extensive study of 742 landraces of all cultivated species (or Cultivar Groups) and 8 closely related wild species progenitors, with 50 nuclear simple sequence repeat (SSR) (also known as microsatellite) primer pairs and a plastid DNA deletion marker that distinguishes most lowland Chilean from upland Andean landraces. Neighbor-joining results highlight a tendency to separate three groups: (i) putative diploids, (ii) putative tetraploids, and (iii) the hybrid cultivated species S. ajanhuiri (diploid), S. juzepczukii (triploid), and S. curtilobum (pentaploid). However, there are many exceptions to grouping by ploidy. Strong statistical support occurs only for S. ajanhuiri, S. juzepczukii, and S. curtilobum. In combination with recent morphological analyses and an examination of the identification history of these collections, we support the reclassification of the cultivated potatoes into four species: (i) S. tuberosum, with two Cultivar Groups (Andigenum Group of upland Andean genotypes containing diploids, triploids, and tetraploids, and the Chilotanum Group of lowland tetraploid Chilean landraces); (ii) S. ajanhuiri (diploid); (iii) S. juzepczukii (triploid); and (iv) S. curtilobum (pentaploid). For other classifications, consistent and stable identifications are impossible, and their classification as species is artificial and only maintains the confusion of users of the gene banks and literature.

Andean potato cultivars (Solanum tuberosum L.) as a source of antioxidant and mineral micronutrients.

Potato tubers were evaluated as a source of antioxidants and minerals for the human diet. A genetically diverse sample of Solanum tuberosum L. cultivars native to the Andes of South America was obtained from a collection of nearly 1000 genotypes using microsatellite markers. This size-manageable collection of 74 landraces, representing at best the genetic diversity among potato germplasm, was analyzed for iron, zinc, calcium, total phenolic, total carotenoid, and total vitamin C contents. The hydrophilic antioxidant capacity of each genotype was also measured using the oxygen radical absorbance capacity (ORAC) assay. The iron content ranged from 29.87 to 157.96 microg g-1 of dry weight (DW), the zinc content from 12.6 to 28.83 microg g-1 of DW, and the calcium content from 271.09 to 1092.93 microg g-1 of DW. Total phenolic content varied between 1.12 and 12.37 mg of gallic acid equiv g-1 of DW, total carotenoid content between 2.83 and 36.21 microg g-1 of DW, and total vitamin C content between 217.70 and 689.47 microg g-1 of DW. The range of hydrophilic ORAC values was 28.25-250.67 micromol of Trolox equiv g-1 of DW. The hydrophilic antioxidant capacity and the total phenolic content were highly and positively correlated (r = 0.91). A strong relationship between iron and calcium contents was also found (r = 0.67). Principal component analysis on the studied nutritional contents of the core collection revealed that most potato genotypes were balanced in terms of antioxidant and mineral contents, but some of them could be distinguished by their high level in distinct micronutrients. Correlations between the micronutrient contents observed in the sample and the genetic distances assessed by microsatellites were weakly significant. However, this study demonstrated the wide variability of health-promoting micronutrient levels within the native potato germplasm as well as the significant contribution that distinct potato tubers may impart to the intake in dietary antioxidants, zinc, and iron.