Comparing the performances of SSR and SNP markers for population analysis in Theobroma cacao L., as alternative approach to validate a new ddRADseq protocol for cacao genotyping.

Proper cacao (Theobroma cacao L.) plant genotyping is mandatory for the conservation and use of the species genetic resources. A set of 15 international standard SSR markers was assumed as universal cacao genotyping system. Recently, different SNPs and SNP genotyping techniques have been exploited in cacao. However, a consensus on which to use has not been reached yet, driving the search for new approaches. To validate a new ddRADseq protocol for cacao genotyping, we compared the performances for population analysis of a dataset with 7,880 SNPs obtained from ddRADseq and the genotypic data from the aforementioned SSR set, using 158 cacao plants from productive farms and gene bank. Four genetic groups were identified with STRUCTURE and ADMIXTURE softwares using SSR and SNP data, respectively. Similarities of cacao ancestries among these groups allowed the identification of analogous pairs of groups of individuals, referred to as: G1SSR/G1SNP, G2SSR/G2SNP, G3SSR/G3SNP, G4SSR/G4SNP, whether SSRs or SNPs were used. Both marker systems identified Amelonado and Criollo as the most abundant cacao ancestries among all samples. Genetic distance matrices from both data types were significantly similar to each other according to Mantel test (p < 0.0001). PCoA and UPGMA clustering mostly confirmed the identified genetic groups. AMOVA and FST pairwise comparison revealed a moderate to very large genetic differentiation among identified groups from SSR and SNP data. Genetic diversity parameters from SSR (Hobs = 0.616, Hexp = 0.524 and PIC = 0.544) were higher than that from SNP data (0.288, 0.264, 0.230). In both cases, genetic groups carrying the highest Amelonado proportion (G1SSR and G1SNP) had the lowest genetic diversity parameters among the identified groups. The high congruence among population analysis results using both systems validated the ddRADseq protocol employed for cacao SNP genotyping. These results could provide new ways for developing a universal SNP-based genotyping system very much needed for cacao genetic studies.

Using ddRADseq to assess the genetic diversity of in-farm and gene bank cacao resources in the Baracoa region, eastern Cuba, for use and conservation purposes.

The Baracoa region, eastern Cuba, hosts around 80 % of the country cacao (Theobroma cacao L.) plantations. Cacao plants in farms are diverse in origin and propagation, with grafted and hybrid plants being the more common ones. Less frequent are plants from cuttings, TSH progeny, and traditional Cuban cacao. A national cacao gene bank is also present in Baracoa, with 282 accessions either prospected in Cuba or introduced from other countries. A breeding program associated with the gene bank started in the 1990s based on agro-morphological descriptors. The genetic diversity of cacao resources in Baracoa has been poorly described, except for traditional Cuban cacao, affecting the proper development of the breeding program and the cacao planting policies in the region. To assess the population structure and genetic diversity of cacao resources in Baracoa region, we genotyped plants from both cacao gene bank (CG) and cacao farms (CF) applying a new ddRADseq protocol for cacao. After data processing, two SNPs datasets containing 11,425 and 6,481 high-quality SNPs were generated with 238 CG and 135 CF plants, respectively. SNPs were unevenly distributed along the 10 cacao chromosomes and laid mainly in noncoding regions of the genome. Population structure analysis with these SNP datasets identified seven and four genetic groups in CG and CF samples, respectively. Clustering using UPGMA and principal component analysis mostly agree with population structure results. Amelonado was the predominant cacao ancestry, accounting for 49.22 % (CG) and 57.73 % (CF) of the total. Criollo, Contamana, Iquitos, and Nanay ancestries were detected in both CG and CF samples, while Nacional and Marañon backgrounds were only identified in CG. Genetic differentiation among CG (FST ranging from 0.071 to 0.407) was higher than among CF genetic groups (FST: 0.093-0.282). Genetic diversity parameters showed similar values for CG and CF samples. The CG and CF genetic groups with the lowest genetic diversity parameters had the highest proportion of Amelonado ancestry. These results should contribute to reinforcing the ongoing breeding program and updating the planting policies on cacao farms, with an impact on the social and economic life of the region.

Integration of genome-wide association studies reveal loci associated with salt tolerance score of rice at the seedling stage.

Salt threatens rice cultivation in many countries. Hence, breeding new varieties with high salt tolerance is important.A panel of 2,391 rice accessions from the 3 K Rice Genome Project was selected to evaluate salt tolerance via the standard evaluation score (SES) in hydroponics under 60 mM NaCl at the seedling stage. Three sub-population panels including 1,332, 628, and 386 accessions from the original 2,391 ones were constructed based on low relatedness revealed by a phylogenetic tree generated by Archaeopteryx Tree. A genome-wide association study (GWAS) was conducted on the entire and sub-population panels using SES data and a selection of 5, 10, 20, and 40% of SNPs selected from the original 1,011,601 SNPs by filtering minor allele frequency > 5% and missing rate < 5%. To perform GWAS, three methods implemented in three different software packages were utilized.Using the integration of GWAS programs, a total of four QTLs associated with SES scores were identified in different panels. Some QTLs co-located with previously detected QTL-related traits. qSES1.1 was detected in three panels, qSES1.3 and qSES2.1 in two panels, and qSES3.1 in one panel through GWAS by all three methods used and selected SNPs. These four QTLs were selected to detect candidate genes. Combining gene-based association study plus haplotype analysis in the entire population and the three sub-populations let us shortlist three candidate genes, viz. LOC_Os01g23640 and LOC_Os01g23680 for qSES1.1, and LOC_Os01g71240 for qSES1.3 region affecting salt tolerance. The identified QTLs and candidate genes provided useful materials and genetic information for future functional characterization and genetic improvement of salt tolerance in rice.

Identification of quantitative trait loci controlling nitrogen use efficiency-related traits in rice at the seedling stage under salt condition by genome-wide association study.

Rice cultivation is facing both salt intrusion and overuse of nitrogen fertilizers. Hence, breeding new varieties aiming to improve nitrogen use efficiency (NUE), especially under salt conditions, is indispensable. We selected 2,391 rice accessions from the 3K Rice Genomes Project to evaluate the dry weight under two N concentrations [2.86 mM - standard N (SN), and 0.36 mM - low N (LN)] crossed with two NaCl concentrations [0 (0Na) and 60 mM (60Na)] at the seedling stage. Genome-wide association studies for shoot, root, and plant dry weight (DW) were carried out. A total of 55 QTLs - 32, 16, and 7 in the whole, indica, and japonica panel - associated with one of the tested traits were identified. Among these, 27 QTLs co-localized with previously identified QTLs for DW-related traits while the other 28 were newly detected; 24, 8, 11, and 4 QTLs were detected in SN-0Na, LN-0Na, SN-60Na, and LN-60Na, respectively, and the remaining 8 QTLs were for the relative plant DW between treatments. Three of the 11 QTLs in SN-60Na were close to the regions containing three QTLs detected in SN-0Na. Eleven candidate genes for eight important QTLs were identified. Only one of them was detected in both SN-0Na and SN-60Na, while 5, 0, 3, and 2 candidate genes were identified only once under SN-0Na, LN-0Na, SN-60Na, and LN-60Na, respectively. The identified QTLs and genes provide useful materials and genetic information for future functional characterization and genetic improvement of NUE in rice, especially under salt conditions.

Changes in bacterial diversity of activated sludge exposed to titanium dioxide nanoparticles.

The rapid growth of the use of nanomaterials in different modern industrial branches makes the study of the impact of nanoparticles on the human health and environment an urgent matter. For instance, it has been reported that titanium dioxide nanoparticles (TiO2 NPs) can be found in wastewater treatment plants. Previous studies have found contrasting effects of these nanoparticles over the activated sludge process, including negative effects on the oxygen uptake. The non-utilization of oxygen reflects that aerobic bacteria were inhibited or decayed. The aim of this work was to study how TiO2 NPs affect the bacterial diversity and metabolic processes on an activated sludge. First, respirometry assays of 8 h were carried out at different concentrations of TiO2 NPs (0.5-2.0 mg/mL) to measure the oxygen uptake by the activated sludge. The bacterial diversity of these assays was determined by sequencing the amplified V3-V4 region of the 16S rRNA gene using Illumina MiSeq. According to the respirometry assays, the aerobic processes were inhibited in a range from 18.5 ± 4.8% to 37.5 ± 2.0% for concentrations of 0.5-2.0 mg/mL TiO2 NPs. The oxygen uptake rate was affected mainly after 4.5 h for concentrations higher than 1.0 mg/mL of these nanoparticles. Results indicated that, in the presence of TiO2 NPs, the bacterial community of activated sludge was altered mainly in the genera related to nitrogen removal (nitrogen assimilation, nitrification and denitrification). The metabolic pathways prediction suggested that genes related to biofilm formation were more sensitive than genes directly related to nitrification-denitrification and N-assimilation processes. These results indicated that TiO2 NPs might modify the bacteria diversity in the activated sludge according to their concentration and time of exposition, which in turn impact in the performance of the wastewater treatment processes.

A Case of Gene Fragmentation in Plant Mitochondria Fixed by the Selection of a Compensatory Restorer of Fertility-Like PPR Gene.

The high mutational load of mitochondrial genomes combined with their uniparental inheritance and high polyploidy favors the maintenance of deleterious mutations within populations. How cells compose and adapt to the accumulation of disadvantageous mitochondrial alleles remains unclear. Most harmful changes are likely corrected by purifying selection, however, the intimate collaboration between mitochondria- and nuclear-encoded gene products offers theoretical potential for compensatory adaptive changes. In plants, cytoplasmic male sterilities are known examples of nucleo-mitochondrial coadaptation situations in which nuclear-encoded restorer of fertility (Rf) genes evolve to counteract the effect of mitochondria-encoded cytoplasmic male sterility (CMS) genes and restore fertility. Most cloned Rfs belong to a small monophyletic group, comprising 26 pentatricopeptide repeat genes in Arabidopsis, called Rf-like (RFL). In this analysis, we explored the functional diversity of RFL genes in Arabidopsis and found that the RFL8 gene is not related to CMS suppression but essential for plant embryo development. In vitro-rescued rfl8 plantlets are deficient in the production of the mitochondrial heme-lyase complex. A complete ensemble of molecular and genetic analyses allowed us to demonstrate that the RFL8 gene has been selected to permit the translation of the mitochondrial ccmFN2 gene encoding a heme-lyase complex subunit which derives from the split of the ccmFN gene, specifically in Brassicaceae plants. This study represents thus a clear case of nuclear compensation to a lineage-specific mitochondrial genomic rearrangement in plants and demonstrates that RFL genes can be selected in response to other mitochondrial deviancies than CMS suppression.

Divergent effects of translation termination factor eRF3A and nonsense-mediated mRNA decay factor UPF1 on the expression of uORF carrying mRNAs and ribosome protein genes.

In addition to its role in translation termination, eRF3A has been implicated in the nonsense-mediated mRNA decay (NMD) pathway through its interaction with UPF1. NMD is a RNA quality control mechanism, which detects and degrades aberrant mRNAs as well as some normal transcripts including those that harbour upstream open reading frames in their 5' leader sequence. In this study, we used RNA-sequencing and ribosome profiling to perform a genome wide analysis of the effect of either eRF3A or UPF1 depletion in human cells. Our bioinformatics analyses allow to delineate the features of the transcripts controlled by eRF3A and UPF1 and to compare the effect of each of these factors on gene expression. We find that eRF3A and UPF1 have very different impacts on the human transcriptome, less than 250 transcripts being targeted by both factors. We show that eRF3A depletion globally derepresses the expression of mRNAs containing translated uORFs while UPF1 knockdown derepresses only the mRNAs harbouring uORFs with an AUG codon in an optimal context for translation initiation. Finally, we also find that eRF3A and UPF1 have opposite effects on ribosome protein gene expression. Together, our results provide important elements for understanding the impact of translation termination and NMD on the human transcriptome and reveal novel determinants of ribosome biogenesis regulation.

The bacterial diversity on steam vents from Paricutín and Sapichu volcanoes.

Vapor steam vents are prevailing structures on geothermal sites in which local geochemical conditions allow the development of extremophilic microorganisms. We describe the structure of the prokaryotic community able to grow on the walls and rocks of such microecosystems in two terrestrial Mexican volcanoes: Paricutín (PI and PII samples) and its satellite Sapichu (S sample). The investigated samples showed similar diversity indices, with few dominant OTUs (abundance > 1%): 21, 16 and 23, respectively for PI, PII and S. However, each steam vent showed a particular community profile: PI was dominated by photosynthetic bacteria (Cyanobacteria and Chloroflexia class), PII by Actinobacteria and Proteobacteria, and S by Ktedonobacteria class, Acidobacteria and Cyanobacteria phyla. Concerning the predicted metabolic potential, we found a dominance of cellular pathways, especially the ones for energy generation with metabolisms for sulfur respiration, nitrogen fixation, methanogenesis, carbon fixation, photosynthesis, and metals, among others. We suggest a different maturity stage for the three studied fumaroles, from the youngest (PI) to the oldest (S and PII), also influenced by the temperature and other geochemical parameters. Furthermore, four anaerobic strains were isolated, belonging to Clostridia class (Clostridium sphenoides, C. swellfunanium and Anaerocolumna cellulosilytica) and to Bacilli class (Paenibacillus azoreducens).

Genetic and environmental factors affecting the expression of α-gliadin canonical epitopes involved in celiac disease in a wide collection of spelt (Triticum aestivum ssp. spelta) cultivars and landraces.

BACKGROUND: Celiac disease (CD) is an autoimmune disorder affecting genetically predisposed individuals whose dietary gluten proteins trigger an inflammatory reaction in the small intestine. Gluten is found in the seeds of cereals like bread wheat (Triticum aestivum ssp. aestivum) and spelt (Triticum aestivum ssp. spelta). The development of new varieties lacking immunogenic peptides is one of the strategies currently investigated to address the CD problem. Among gluten proteins, α-gliadins display the strongest immunogenicity with four main T-cell stimulatory epitopes. The objective of this work was to study the expression of α-gliadin epitopes related to CD in a wide collection of 121 spelt accessions (landraces and varieties, spring and winter accessions) from different provenances, and to analyze the correlation between the presence of epitope sequences in gDNA and their expression (cDNA). The effect of environmental factors (harvest year and N fertilization) on the epitope expression was also investigated. RESULTS: TaqMan probes targeting the canonical form of the epitopes were used to evaluate the epitope expression levels. Significant variations in the amount of epitope transcripts were identified between accessions and according to the provenances. Spring accessions showed a significantly higher immunogenicity than winter ones and no influence of spelt breeding on the epitope expression levels could be assessed when comparing landraces and varieties from Northwestern Europe. No correlation was observed between quantitative PCR results obtained from cDNA and gDNA for 45 accessions tested, stressing the need to use markers focusing on epitope transcripts rather than on genomic sequences. A relative stability of the amount of epitopes expressed by a same accession across four harvest years was detected. The fertilization strategy, evaluated through seven N fertilization modalities applied to two commercial spelt varieties, did not influence the epitope expression of the first variety, whereas it had a slight effect for the second one. CONCLUSIONS: The results obtained in this work showed that the CD-related epitope expression greatly fluctuated among the spelt accessions studied. This expression was not correlated to the epitope genomic occurrence and environmental factors had almost no influence on the amount of epitope transcripts.

Reactivity of gluten proteins from spelt and bread wheat accessions towards A1 and G12 antibodies in the framework of celiac disease.

In the framework of celiac disease, this research aims at evaluating the reactivity of 195 wheat accessions and 240 spelt accessions to A1 and G12 monoclonal antibodies. A great variability in reactivity was found among the accessions of both subspecies. On average, spelt showed very slightly higher reactivity than wheat but accessions with low reactivity were encountered in both subspecies. In both wheat and spelt, there was no significant difference in the level of reactivity between varieties and landraces. Similarly, there was no significant difference in reactivity between old, mid and new varieties of wheat. In contrast, new spelt varieties showed lower levels of reactivity than old and mid ones. No relationship could be established between level of reactivity, protein content and the Zeleny index. This research did not establish a link between the breeding strategies for baking quality improvement and A1-G12 antibodies reactivity.

The translational landscape of Arabidopsis mitochondria.

Messenger RNA translation is a complex process that is still poorly understood in eukaryotic organelles like mitochondria. Growing evidence indicates though that mitochondrial translation differs from its bacterial counterpart in many key aspects. In this analysis, we have used ribosome profiling technology to generate a genome-wide snapshot view of mitochondrial translation in Arabidopsis. We show that, unlike in humans, most Arabidopsis mitochondrial ribosome footprints measure 27 and 28 bases. We also reveal that respiratory subunits encoding mRNAs show much higher ribosome association than other mitochondrial mRNAs, implying that they are translated at higher levels. Homogenous ribosome densities were generally detected within each respiratory complex except for complex V, where higher ribosome coverage corroborated with higher requirements for specific subunits. In complex I respiratory mutants, a reorganization of mitochondrial mRNAs ribosome association was detected involving increased ribosome densities for certain ribosomal protein encoding transcripts and a reduction in translation of a few complex V mRNAs. Taken together, our observations reveal that plant mitochondrial translation is a dynamic process and that translational control is important for gene expression in plant mitochondria. This study paves the way for future advances in the understanding translation in higher plant mitochondria.

Deciphering the reading of the genetic code by near-cognate tRNA.

Some codons of the genetic code can be read not only by cognate, but also by near-cognate tRNAs. This flexibility is thought to be conferred mainly by a mismatch between the third base of the codon and the first of the anticodon (the so-called "wobble" position). However, this simplistic explanation underestimates the importance of nucleotide modifications in the decoding process. Using a system in which only near-cognate tRNAs can decode a specific codon, we investigated the role of six modifications of the anticodon, or adjacent nucleotides, of the tRNAs specific for Tyr, Gln, Lys, Trp, Cys, and Arg in Saccharomyces cerevisiae. Modifications almost systematically rendered these tRNAs able to act as near-cognate tRNAs at stop codons, even though they involve noncanonical base pairs, without markedly affecting their ability to decode cognate or near-cognate sense codons. These findings reveal an important effect of modifications to tRNA decoding with implications for understanding the flexibility of the genetic code.

Evidence for rRNA 2'-O-methylation plasticity: Control of intrinsic translational capabilities of human ribosomes.

Ribosomal RNAs (rRNAs) are main effectors of messenger RNA (mRNA) decoding, peptide-bond formation, and ribosome dynamics during translation. Ribose 2'-O-methylation (2'-O-Me) is the most abundant rRNA chemical modification, and displays a complex pattern in rRNA. 2'-O-Me was shown to be essential for accurate and efficient protein synthesis in eukaryotic cells. However, whether rRNA 2'-O-Me is an adjustable feature of the human ribosome and a means of regulating ribosome function remains to be determined. Here we challenged rRNA 2'-O-Me globally by inhibiting the rRNA methyl-transferase fibrillarin in human cells. Using RiboMethSeq, a nonbiased quantitative mapping of 2'-O-Me, we identified a repertoire of 2'-O-Me sites subjected to variation and demonstrate that functional domains of ribosomes are targets of 2'-O-Me plasticity. Using the cricket paralysis virus internal ribosome entry site element, coupled to in vitro translation, we show that the intrinsic capability of ribosomes to translate mRNAs is modulated through a 2'-O-Me pattern and not by nonribosomal actors of the translational machinery. Our data establish rRNA 2'-O-Me plasticity as a mechanism providing functional specificity to human ribosomes.

Development of TaqMan probes targeting the four major celiac disease epitopes found in α-gliadin sequences of spelt (Triticum aestivum ssp. spelta) and bread wheat (Triticum aestivum ssp. aestivum).

BACKGROUND: Celiac disease (CD) is caused by specific sequences of gluten proteins found in cereals such as bread wheat (Triticum aestivum ssp. aestivum) and spelt (T. aestivum ssp. spelta). Among them, the α-gliadins display the highest immunogenicity, with four T-cell stimulatory epitopes. The toxicity of each epitope sequence can be reduced or even suppressed according to the allelic form of each sequence. One way to address the CD problem would be to make use of this allelic variability in breeding programs to develop safe varieties, but tools to track the presence of toxic epitopes are required. The objective of this study was to develop a tool to accurately detect and quantify the immunogenic content of expressed α-gliadins of spelt and bread wheat. RESULTS: Four TaqMan probes that only hybridize to the canonical-i.e. toxic-form of each of the four epitopes were developed and their specificity was demonstrated. Six TaqMan probes targeting stable reference genes were also developed and constitute a tool to normalize qPCR data. The probes were used to measure the epitope expression levels of 11 contrasted spelt accessions and three ancestral diploid accessions of bread wheat and spelt. A high expression variability was highlighted among epitopes and among accessions, especially in Asian spelts, which showed lower epitope expression levels than the other spelts. Some discrepancies were identified between the canonical epitope expression level and the global amount of expressed α-gliadins, which makes the designed TaqMan probes a useful tool to quantify the immunogenic potential independently of the global amount of expressed α-gliadins. CONCLUSIONS: The results obtained in this study provide useful tools to study the immunogenic potential of expressed α-gliadin sequences from Triticeae accessions such as spelt and bread wheat. The application of the designed probes to contrasted spelt accessions revealed a high variability and interesting low canonical epitope expression levels in the Asian spelt accessions studied.

Molecular diversity of α-gliadin expressed genes in genetically contrasted spelt (Triticum aestivum ssp. spelta) accessions and comparison with bread wheat (T. aestivum ssp. aestivum) and related diploid Triticum and Aegilops species.

The gluten proteins of cereals such as bread wheat (Triticum aestivum ssp. aestivum) and spelt (T. aestivum ssp. spelta) are responsible for celiac disease (CD). The α-gliadins constitute the most immunogenic class of gluten proteins as they include four main T-cell stimulatory epitopes that affect CD patients. Spelt has been less studied than bread wheat and could constitute a source of valuable diversity. The objective of this work was to study the genetic diversity of spelt α-gliadin transcripts and to compare it with those of bread wheat. Genotyping data from 85 spelt accessions obtained with 19 simple sequence repeat (SSR) markers were used to select 11 contrasted accessions, from which 446 full open reading frame α-gliadin genes were cloned and sequenced, which revealed a high allelic diversity. High variations among the accessions were highlighted, in terms of the proportion of α-gliadin sequences from each of the three genomes (A, B and D), and their composition in the four T-cell stimulatory epitopes. An accession from Tajikistan stood out, having a particularly high proportion of α-gliadins from the B genome and a low immunogenic content. Even if no clear separation between spelt and bread wheat sequences was shown, spelt α-gliadins displayed specific features concerning e.g. the frequencies of some amino acid substitutions. Given this observation and the variations in toxicity revealed in the spelt accessions in this study, the high genetic diversity held in spelt germplasm collections could be a valuable resource in the development of safer varieties for CD patients.

Mutations in chicory FEH genes are statistically associated with enhanced resistance to post-harvest inulin depolymerization.

KEY MESSAGE: Nucleotidic polymorphisms were identified in fructan exohydrolases genes which are statistically associated with enhanced susceptibility to post-harvest inulin depolymerization. Industrial chicory (Cichorium intybus L.) root is the main commercial source of inulin, a linear fructose polymer used as dietary fiber. Post-harvest, inulin is depolymerized into fructose which drastically increases processing cost. To identify genetic variations associated with enhanced susceptibility to post-harvest inulin depolymerization and related free sugars content increase, we used a candidate-gene approach focused on inulin and sucrose synthesis and degradation genes, all members of the family 32 of glycoside hydrolases (GH32). Polymorphism in these genes was first investigated by carrying out EcoTILLING on two groups of chicory breeding lines exhibiting contrasted response to post-harvest inulin depolymerization. This allowed the identification of polymorphisms significantly associated with depolymerization in three fructan exohydrolase genes (FEH). This association was confirmed on a wider panel of 116 unrelated families in which the FEH polymorphism explained 35 % of the post-harvest variance for inulin content, 36 % of variance for sucrose content, 18 % for inulin degree of polymerization, 23 % for free fructose content and 22 % for free glucose content. These polymorphisms were associated with significant post-harvest changes of inulin content, inulin chain length and free sugars content.

Combined transcriptomic and physiological approaches reveal strong differences between short- and long-term response of rice (Oryza sativa) to iron toxicity.

Ferrous iron toxicity is a mineral disorder frequently occurring under waterlogged soils where rice is cultivated. To decipher the main metabolic pathways involved in rice response to iron excess, seedlings have been exposed to 125 mg L(-1) FeSO(4) for 3 weeks. A combined transcriptomic, biochemical and physiological study has been performed after short-term (3 d) or long-term (3 weeks) exposure to iron in order to elucidate the strategy of stress adaptation with time. Our results showed that short- and long-term exposure involved a very different response in gene expression regarding both the number and function. A larger number of genes were up- or down-regulated after 3 d than after 3 weeks of iron treatment; these changes also occurred in shoot even though no significant difference in iron concentration was recorded. Those modifications in gene expression after 3 d affected not only genes involved in hormonal signalling but also genes involved in C-compound and carbohydrate metabolism, oxygen and electron transfer, oxidative stress, and iron homeostasis and transport. Modification in some gene expression can be followed by modification in corresponding metabolic products and physiological properties, or differed in time for some others, underlying the importance of an integrated study.

Reproductive biology of the andromonoecious Cucumis melo subsp. agrestis (Cucurbitaceae).

BACKGROUND AND AIMS: Cucumis melo subsp. agrestis (Cucurbitaceae) is cultivated in many African regions for its edible kernels used as a soup thickener. The plant, an annual, andromonoecious, trailing-vine species, is of high social, cultural and economic value for local communities. In order to improve the yield of this crop, the first step and our aim were to elucidate its breeding system. METHODS: Eight experimental pollination treatments were performed during three growing seasons to assess spontaneous selfing, self-compatibility and effects of pollen source (hermaphroditic vs. male flowers). Pollination success was determined by pollen tube growth and reproductive success was assessed by fruit, seed and seedling numbers and characteristics. The pollinator guild was surveyed and the pollination distance determined both by direct observations and by indirect fluorescent dye dispersal. KEY RESULTS: The species is probably pollinated by several Hymenoptera, principally by Hypotrigona para. Pollinator flight distances varied from 25 to 69 cm. No evidence for apomixis or spontaneous self-pollination in the absence of insect visitors was found. The self-fertility index (SFI = 0) indicated a total dependence on pollinators for reproductive success. The effects of hand pollination on fruit set, seed number and seedling fitness differed among years. Pollen tube growth and reproductive success did not differ between self- and cross-pollinations. Accordingly, a high self-compatibility index for the fruit set (SCI = 1.00) and the seed number (SCI = 0.98) and a low inbreeding depression at all developmental stages (cumulative delta = 0.126) suggest a high selfing ability. Finally, pollen origin had no effect on fruit and seed sets. CONCLUSIONS: This andromonoecious species has the potential for a mixed mating system with high dependence on insect-mediated pollination. The selfing rate through geitonogamy should be important.

Congruence between morphological and molecular markers inferred from the analysis of the intra-morphotype genetic diversity and the spatial structure of Oxalis tuberosa Mol.

Oxalis tuberosa is an important crop cultivated in the highest Andean zones. A germplasm collection is maintained ex situ by CIP, which has developed a morphological markers system to classify the accessions into morphotypes, i.e. groups of morphologically identical accessions. However, their genetic uniformity is currently unknown. The ISSR technique was used in two experiments to determine the relationships between both morphological and molecular markers systems. The intra-morphotype genetic diversity, the spatial structures of the diversity and the congruence between both markers systems were determined. In the first experience, 44 accessions representing five morphotypes, clearly distinct from each other, were analyzed. At the molecular level, the accessions exactly clustered according to their morphotypes. However, a genetic variability was observed inside each morphotype. In the second experiment, 34 accessions gradually differing from each other on morphological base were analyzed. The morphological clustering showed no geographical structure. On the opposite, the molecular analysis showed that the genetic structure was slightly related to the collection site. The correlation between both markers systems was weak but significant. The lack of perfect congruence between morphological and molecular data suggests that the morphological system may be useful for the morphotypes management but is not appropriate to study the genetic structure of the oca. The spatial structure of the genetic diversity can be related to the evolution of the species and the discordance between the morphological and molecular structures may result from similar selection pressures at different places leading to similar forms with a different genetic background.

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.