Pierre Bertin

Genetic diversity among European cultivated spelt revealed by microsatellites

Abstract Spelt and common wheat constitute two of the six groups of the hexaploid wheats with an AABBDD genome. Spelt culture has been progressively replaced by that of common wheat which out-yields spelt under high-input conditions. In the last decades, spelt breeders intended to introduce the yield-potential and bread-making qualities of common wheat into spelt, by frequent crossings between accessions of these two different groups. The present study aims at determining the genetic basis of modern spelt cultivars in terms of intra-group variability and inter-group (spelt vs common wheat) distances, by using microsatellite markers developed for common wheat. The allelic composition of 30 spelt and nine common wheat accessions was determined at 17 microsatellite loci. The coefficient of co-ancestry (ƒ) and the genetic distances (1 - proportion of shared alleles) based upon allelic composition were calculated for all pairs of accessions. Two dendrograms were constructed using the UPGMA method. Amplification products were found for all loci on most accessions. A total of 113 alleles was identified, of which 60.2% were specific to spelt or common wheat. The correlation between (1 –ƒ ) and the genetic distance was high (0.701***). The mean pairwise genetic distance was 0.656 ± 0.181 over the 39 accessions, 0.706 ± 0.14 among common wheat and 0.573 ± 0.172 among spelt. The mean genetic distance between spelt and wheat was 0.782 ± 0.113. The two dendrograms were in accordance with each other and clearly separated the spelt from the common wheat accessions. It is concluded that microsatellites developed for common wheat and distances based on the proportion of shared alleles are powerful tools for reconstructing phylogenies in spelt, and that the genetic basis of modern spelt cultivars is narrow despite frequent crosses made with bread wheat.

Evaluation of chilling sensitivity in different rice varieties. Relationship between screening procedures applied during germination and vegetative growth

SummaryRice varieties were screened for chilling tolerance during germination and vegetative growth using different techniques. The effects of temperature during germination were investigated from 10 to 25°C. The screening was most effective at 10°C. Time of data collection has to be considered in order to discriminate slow germinating from chilling sensitive varieties. Chilling survival tests (10°C) applied at the 2-leaf stage revealed that different chilling durations brought complementary results: the longer treatments allowed to identify the most tolerant varieties, while shorter treatments allowed to distinguish intermediate from most sensitive rices. Results of chilling tests applied at the 2- and the 8-leaf stages showed a high correlation. Low-temperature electrolyte leakage and low-temperature fluorescence were also highly correlated to each other and to survival tests applied at the same development stage. By contrast, weak correlations were observed between low-temperature germination and plantlet chilling survival. It is concluded that low-temperature electrolyte leakage and low-temperature fluorescence are good indicators of chilling tolerance during the vegetative growth. The possibility to use a chilling screening procedure applied at this stage in order to predict the chilling tolerance at other development stages is discussed.

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.

Evaluation of drought resistance-related traits in durum wheat somaclonal lines selected in vitro

Somaclonal variation associated with in vitro selection has been used as a source of variability to improve drought resistance of 3 durum wheat (Triticum durum Desf.) cultivars (Selbera, Sebou, and Kyperounda). In a previous study, R0 plants with improved drought resistance-related characters were regenerated after selection on culture media containing polyethylene glycol (PEG). This improvement was transmitted to the R1 progeny. The present study analysed the behaviour of the selected tissue culture-derived lines in subsequent R2, R3 and R4 generations. Differences in electrolyte leakage, chlorophyll fluorescence (F-v/F-m), stomatal conductance and days to heading were found between the parental cultivars and most of their in vitro-derived lines. The changes may differ from one cultivar to another. Many promising somaclonal lines still presented improvement for at least 3 of the 4 parameters measured comparatively to initial cultivars. Somaclonal variation thus appears to induce a wide range of modifications among individual components of drought-resistance mechanisms. These improved traits could be valuable if shown to be inherited and to give enhanced agronomic performances in future field studies.

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.

Use of somaclonal variation and in vitro selection for chilling tolerance improvement in rice

Embryo-derived calli of four rice varieties cultivated at high altitude in Burundi — Facagro 57, Facagro 76, Kirundo 3 and Kirundo 9 — were submitted to different temperature regimes. The percentage of regenerating calli greatly varied depending on variety, length of culture and callus temperature treatment. The reduction of regeneration percentages induced by low temperature was more pronounced in the more sensitive varieties. Regenerated plants (R0) and their progenies in R1, R2 and R3 were cold-screened together with control plants. In all varieties, significantly higher survival rates were obtained in R3 with in vitro plants than with control plants. Such chilling tolerance improvement was not obtained following a massal selection applied during 3 successive generations onto the control plants. In vitro plants regenerated from calli cultivated either at 25 °C, either at 4 °C, were cultivated at different altitudes in Burundi during two successive generations. For most observed traits, the in vitro plants were characterized by lower means, larger variation and higher maximum values than the control plants. The most chilling-tolerant somaclonal families were most usually characterized by extensive differences in fatty acid composition, chilling-induced electrolyte leakage and chlorophyll fluorescence, compared to the varieties they derived from.

Set up of simple sequence repeat markers and first investigation of the genetic diversity of West-African watermelon (Citrullus lanatus ssp. vulgaris oleaginous type)

Citrullus lanatus ssp. vulgaris oleaginous type (West-African watermelon) is a crop cultivated in sub-Saharan Africa for its dried seeds reported to be rich in nutrients. In previous studies, little polymorphism was found in watermelon—cultivated for its flesh with the use of microsatellite (SSR) markers. Such study has never been applied to the oleaginous type until now. The objectives of the present study were firstly to apply the SSR markers set up for watermelon to the West-African watermelon and secondly to study the genetic structure of this type in Ivory Coast. For the first objective, 37 markers were studied on eight plants pertaining to four accessions. For the second objective, the polymorphic markers were applied on three morphologically and geographically separated accessions with twenty plants per accession. Multiple correspondence analysis (MCA), unweighted pair-group method with arithmetic averaging (UPGMA), molecular analysis of variance (AMOVA) and assignments test structure were applied. The optimal annealing temperature varied from 49 to 59°C according to the markers. Thirty-two markers that proved to amplify their respective loci were selected, but only nine of them appeared to show polymorphism on the set of 8 plants studied. The application of these markers on the three accessions revealed several features. No stucturation into sub-populations was observed inside a given accession. The genetic variance proved to be substantially higher between the different accessions than inside a given accession. Moreover this analysis is a first hint that the morphology classification does not match the genetic structure of C. lanatus. The results of this work provide the first quantitative information regarding the genetic variability of Citrullus lanatus oleaginous type. In order to sharpen our understanding of the mechanisms responsible for the genetic variance inter/intra accessions, further studies based on a larger sample of plants and accessions are required.

Evidence of intra-varietal genetic variability in the vegetatively propagated crop oca (Oxalis tuberosa Mol.) in the Andean traditional farming system

Oxalis tuberosa is a vegetatively propagated tuber crop in the Andes. The peasants cultivate a great number of varieties for which genetic homogeneity has never been demonstrated. Morphological descriptors and ISSR markers were used to determine the intra-varietal diversity and the influence of the mode of conservation ex-situ vs in-situ. Molecular markers revealed an intra-varietal genetic diversity attesting that oca varieties are not pure clones. The morphological analysis was congruent with the peasant classification, contrary to the molecular markers. The comparison between both conservation strategies revealed a larger intra-varietal diversity in in-situ conditions and a genetic divergence between plants. The traditional practices are likely to be responsible of the intra-varietal polymorphism since the oca is propagated almost exclusively vegetatively. At the conservation level, differences could be explained by the sampling methods. A more integrated approach between genebanks and in-situ conservation is recommended to maintain the genetic resources of the species.

Somaclonal variation and chilling tolerance improvement in rice: changes in fatty acid composition

The relationship between chilling tolerance of six rice cultivars – Facagro 57, Facagro 76, Fujisaka 5, Kirundo 3, Kirundo 9 and IR64 -and the fatty acid composition in total lipids, phospholipids, galactolipids and neutral lipids from leaves was studied. Higher double bond index and proportions of linolenic acid in the phospholipid and galactolipid classes were related to cultivar chilling tolerance, but this was not so for the total lipids nor the neutral lipid class. The somaclonal families derived from Facagro 76, Kirundo 3 and Kirundo 9 that showed enhanced chilling tolerance as compared to their original parental cultivar were analyzed for fatty acid composition in phospholipids and galactolipids from leaves. Altered proportions in fatty acid composition in phospholipids, galactolipids or both were found in the somaclonal families derived from Facagro 76 and Kirundo 9, but not from Kirundo 3. These changes most usually resulted in higher double bond index and higher proportions in linoleic and linolenic acids which were related either to lower ratio of C16 to C18 fatty acids or to higher unsaturation in the C18 fatty acid fraction. Different mechanisms thus seem to be implicated in the altered fatty acid composition of somaclones, which may be related to the chilling tolerance improvement of some somaclonal families.

Genetic diversity of bean (Phaseolus) landraces and wild relatives from the primary centre of origin of the Southern Andes

The Southern Andes, especially the inter-Andean valleys of south Bolivia, is thought to be a probable point of domestication within the primary centre of diversity for Andean common beans (Phaseolus vulgaris L.). The national Phaseolus germplasm collection of Bolivia is maintained by the Pairumani Foundation and consists of 449 accessions where most of the accessions are of common bean but some are of related cultivated and wild species. The goal of this study was to determine the genetic diversity of this collection by sampling 174 accessions of P. vulgaris and an outgroup of eight Phaseolus augusti, two Phaseolus lunatus and one Phaseolus coccineus genotype. The genetic diversity and population structure were estimated using 29 microsatellite markers. High levels of polymorphism were found, with a total of 311 alleles identified and an average of 10.7 alleles per marker. Correspondence analysis and an unweighted pair group method with arithmetic mean-based dendrogram distinguished P. vulgaris from the other species of Phaseolus. Common bean accessions were separated into two groups: the first one including Andean controls and most accessions from high altitudes with morphological characteristics and growth habits typical of this gene pool; the second one including Mesoamerican controls and accessions from low altitudes. Inside the Andean gene pool, the wild accessions were diverse and separated from the weedy and cultivated accessions. Low geographical distances between collection sites (up to 100km) were shown to be related to low genetic distances. These results are important for the conservation of common beans in the Southern Andes.