Exploration of intra- and inter-population genetic diversity in <Emphasis Type="Italic">Hedysarum coronarium</Emphasis> L. by AFLP markers

Amplified fragment length polymorphism (AFLP) markers were used to characterize the genetic diversity within and among natural populations and cultivars of Hedysarum coronarium. Twelve populations within Tunisia were evaluated with three AFLP primer combinations. A total of 207 reproducible bands was detected of which 178 (86%) were polymorphic. The great discriminative power of AFLP markers and their ability to represent genetic relationships among Hedysarum plants was demonstrated. Genetic diversity within and among populations was assessed through Principal Component Analysis (PCA) and cluster analysis by using the Neighbor-joining clustering algorithm. AFLP technology has provided evidence of a high degree of intra- and inter-population genetic diversity in H. coronarium. AFLP banding patterns provided molecular markers correlated with the plants’ geotropism. In addition, AFLP markers can differentiate wild accessions from cultivars. Moreover, geographical origins did not correspond to population clustering.     

<Emphasis Type="Italic">Calamintha nepeta</Emphasis> (L.) Savi and <Emphasis Type="Italic">Micromeria thymifolia</Emphasis> (Scop.) Fritsch cultivated in Italy

Calamintha nepeta and Micromeria thymifolia have been traditionally used in the Mediterranean area as condiments and medicinal plants for a long time. Whereas in parts of Italy C. nepeta (special recipes have been developed in Lazio and Tuscany) is also an established garden plant showing different evolutionary products and their interaction among each other and the wild progenitor, M. thymifolia is being developed into a new crop plant. Both plants and their uses are described with regard to Italy. There is a marked tendency to broaden the use of condiments and spices which results in new crop plants which have to be documented and elaborated in further studies. Many species of Labiatae are predisposed to use by man and new items can be found even in areas which have to be considered as well studied.     

Assessment of genetic variation in common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.) from Nebrodi mountains (Sicily,Italy)

This study was carried out to estimate the level of diversity existing within some common bean landraces still cultivated in Nebrodi mountains, North-western area of Sicily. The multidisciplinary approach adopted to reach this goal involved the characterisation of collected material through morphological, biochemical and molecular marker analyses. The nutritional quality of seeds was also investigated in view of the proposition of the best landraces as niche products. Results showed that those bean landraces retain a considerable level of heterogeneity. The use of both biochemical and molecular markers showed that all landraces clustered into two main groups, corresponding to the Andean and Mesoamerican gene pools. Our results suggest that the best strategy for preserving the diversity of common bean from a restricted area such as Nebrodi mountains, necessitates of a deep knowledge of germplasm to avoid the loss of precious genetic resources or, on the contrary, the safeguard of populations genetically redundant.     

<Emphasis Type="Italic">Ziziphus </Emphasis><Emphasis Type="Italic">spina-christi</Emphasis> (L.) Willd.: a multipurpose fruit tree

Neglected and underutilized species often play a vital role in securing food and livestock feed, income generation and energy needs of rural populations. In spite of their great potential little attention has been given to these species. This increases the possibility of genetic erosion which would further restrict the survival strategies of people in rural areas. Ziziphus spina-christi is a plant species that has edible fruits and a number of other beneficial applications that include the use of leaves as fodder, branches for fencing, wood as fuel, for construction and furniture making, and the utilization of different parts e.g. Fruits, leaves, roots and bark in folk medicine. Moreover, the plant is adapted to dry and hot climates which make it suitable for cultivation in an environment characterized by increasing degradation of land and water resources. Lack of research in Z. spina-christi hinders its successful improvement and promotion. Therefore, studies are needed to fully exploit this species. This article aims at summarizing information on different aspects of Z. spina-christi to stimulate interest in this crop which is of importance in Sudan and other countries of the semi-arid tropics.

Agromorphological diversity of local Bambara groundnut (<Emphasis Type="Italic">Vigna subterranea</Emphasis> (L.) Verdc.) collected in Benin

Bambara groundnut (Vigna subterranea (L.) Verdc.) is a neglected and less exploited species in Benin. However, the rational use of the different landraces available involves an appropriate knowledge of their characteristics. This study examined the characteristics of 52 local landraces of Bambara groundnut traditionally cultivated in Benin based on 12 qualitative and 11 quantitative variables, in order to expose those of good qualities. After a classification based on the morphological variables of the seeds, the experimental field was installed in a completely randomized device of blocks repeated three times. The collected data was analyzed with Statistica 7.1 and NTSYS PC 2.2. softwares using qualitative characteristics (Growth’s type, Stem’s hairiness, Flower’s colour, Leave’s shape etc.). As result, the number of morphotypes increased from 18 (out of classification) to 47. Therefore, it appears that the Benin collection of Bambara groundnut is broad in morphological variability. The analysis of the principal component of the quantitative variables revealed three specific classes within the collection. The most efficient variety which belonged to the second class, had a high field yield per hectare (> 1 t/ha). The Souhouloumankpa variety yielded the earliest (103 days after sowing) in addition to the high field yield. The study also showed a positive relationship between 100 seed’s weight, their length, the number of seeds per plant and the seed’s field yield in kg/ha. For a sustainable preservation and an improvement of the available resources, a molecular characterization of the Bambara groundnut landraces in Benin Republic needs to be done.     

<Emphasis Type="Italic">In situ</Emphasis> conservation and genetic diversity of three populations of <Emphasis Type="Italic">Gossypium mustelinum</Emphasis> Miers ex Watt

Gossypium mustelinum Miers ex Watt is a native from Northeastern Brazil and belongs to the primary gene pool of the cultivated cotton. The unique places where the species was known to occur were visited to plan ex situ and in situ preservation. Two populations at Caicó became extinct, and only eleven individuals were found. At Jaguarari, although one population became extinct, a new one was localized with approximately 500 adult plants, where a fence avoided cattle feeding. The population from Macururé consisted of 28 plants protected from animals by thorns of Bromelia laciniosa Mart. ex Schult. f. Thirteen SSR primer pairs amplified 40 alleles, among which 30% were exclusive of one of the populations. The genetic differences between populations represented 58.3% of the total genetic variation observed. The high genetic distances are likely to be caused by geographical isolation as well as by the small number of individuals which contribute to new generations, and consequential genetic drift.     

Joint toxicity of three plant protection products to <Emphasis Type="Italic">Triticum aestivum</Emphasis> (L.) and <Emphasis Type="Italic">Brassica rapa</Emphasis> (L.)

Purpose  

Few studies have been conducted to evaluate the effects of mixtures of chemicals in terrestrial environment. Thus, it seems important to evaluate if the combined application of pesticides currently used in agricultural fields may pose a risk to terrestrial plants.     

Exploitation of <Emphasis Type="Italic">Malus</Emphasis> EST-SSRs and the utility in evaluation of genetic diversity in <Emphasis Type="Italic">Malus</Emphasis> and <Emphasis Type="Italic">Pyrus</Emphasis>

A total of 8117 suitable SSR-contaning ESTs were acquired by screening from a Malus EST database, among which dinudeotide SSRs were the most abundant repeat motif, within which, CT/TC followed by AG/GA were predominant. Based on the suitable sequences, we developed 147 SSR primer pairs, of which 94 pairs gave amplifications within the expected size range while 65 pairs were found to be polymorphic after a preliminary test. Eighteen primer pairs selected randomly were further used to assess genetic relationship among 20 Malus species or cultivars. As a result, these primers displayed high level of polymorphism with a mean of 6.94 alleles per locus and UPGMA cluster analysis grouped twenty Malus accessions into five groups at the similarity level of 0.6800 that were largely congruent to the traditional taxonomy. Subsequently, all of the 94 primer pairs were tested on four accessions of Pyrus to evaluate the transferability of the markers, and 40 of 72 functional SSRs produced polymorphic amplicons from which 8 SSR loci selected randomly were employed to analyze genetic diversity and relationship among a collection of Pyrus. The 8 primer pairs produced expected bands with the similar size in apples with an average of 7.375 alleles per locus. The observed heterozygosity of different loci ranged from 0.29 (MES₉₆) to 0.83 (MES₁₃₈), with a mean of 0.55 which is lower than 0.63 reported in genome-derived SSR marker analysis in Pyrus. The UPGMA dendrogram was similar to the previous results obtained by using RAPD and AFLP markers. Our results showed that these EST-SSR markers displayed reliable amplification and considerable polymorphism in both Malus and Pyrus, and will contribute to the knowledge of genetic study of Malus and genetically closed genera.     

Comparison of the genetic diversity between <Emphasis Type="Italic">Triticum aestivum</Emphasis> ssp. <Emphasis Type="Italic">tibetanum</Emphasis> Shao and Tibetan wheat landraces (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) by using intron-splice junction primers

In order to evaluate and compare the germplasm resources of wheat in Tibet, we analyzed the genetic diversity of 136 Triticum aestivum ssp. tibetanum Shao and 119 Tibetan wheat landraces (Triticum aestivum L.) by using Intron-Splice Junction (ISJ) primers. The results showed that polymorphism of PCR products were obtained by 33 primer combinations, which accounted for 11% of the 300 primer combinations produced by 26 ISJ primers. A total of 333 stable bands can be amplified from the T. aestivum ssp. tibetanum Shao and 243 bands were polymorphic, which accounted for 72.9% of the total bands. Tibetan wheat Landraces produced 316 stable bands, of which 197 bands were polymorphic. The polymorphic bands accounted for 62.34% of the total bands produced from Tibetan wheat landraces. The genetic diversity of T. aestivum ssp. tibetanum Shao was higher than that of Tibetan wheat landraces in Tibet, suggesting that T. aestivum ssp. tibetanum Shao can be used as important genetic resource for the breeding and genetic improvement of wheat in Tibet. Matrix (1, 0) was generated according to the presence or absence of the bands produced from a particular wheat accession. Clustering and principle coordinates analysis showed that T. aestivum ssp. tibetanum Shao and Tibetan wheat landraces were divided into two groups. We conclude that high polymorphisms produced by ISJ primers can reflect the genetic diversity between T. aestivum ssp. tibetanum Shao and Tibetan wheat landraces.     

Assessment of genetic diversity and relationships among <Emphasis Type="Italic">Triticum urartu</Emphasis> and <Emphasis Type="Italic">Triticum boeoticum</Emphasis> populations from Iran using IRAP and REMAP markers

Einkorn wheat is known as the donor of ‘A’ genome to cultivated wheat and source of many important genes. Therefore, genetic erosion in cultivated wheat provides a good reason to investigate genetic diversity in these species. In the present study, genetic diversity of 14 populations of Triticum urartu and Triticum boeoticum collected from west and north-west of Iran was examined by IRAP and REMAP markers. In total, 26 out of 36 IRAP and 41 out of 88 REMAP combinations amplified polymorphic and scorable banding patterns. IRAP and REMAP combinations produced 6.53 and 5.21 polymorphic bands per assay, respectively. Mean of polymorphism information content for IRAPs and REMAPs were 0.38 and 0.40 and marker index values for them were 2.60 and 2.09, respectively. Analysis of molecular variance based on IRAP and REMAP data revealed significant within and among population variances, although within population variance was higher than that of among population. Primer combinations based on Sukkula and Nikita retrotransposons produced the highest number of markers in the whole population. Cluster and principal coordinate analyses using REMAP data grouped the populations based on the species and geographical origin, but grouping based on IRAP could not separate the two species. However, based on both marker systems considerable diversity was observed among and within the studied populations.     

Genetic diversity of European pear cultivars (<Emphasis Type="Italic">Pyrus communis</Emphasis> L.) and wild pear (<Emphasis Type="Italic">Pyrus pyraster</Emphasis> (L.) Burgsd.) inferred from microsatellite markers analysis

The aim of this study was to identify the group of highly polymorphic microsatellite markers for the identification of six pear cultivars (P. communis) and two individuals of wild pear (P. pyraster). From among 40 tested SSR markers, 19 were selected to profile genetic diversity in pear genotypes due to high polymorphisms. These markers showed high heterozygosity levels (0.5–1) and, on average, 6.4 alleles per marker were found. The set of microsatellite markers employed in this study demonstrated usefulness of microsatellite markers for the identification of pear genotypes. The examined wild forms were represented in this study by only two individuals of P. pyraster. It can be assumed that these forms were distinctly different from the cultivated pear cultivars.     

AFLP analysis of genetic diversity in leafy kale (<Emphasis Type="Italic">Brassica oleracea</Emphasis> L. convar. <Emphasis Type="Italic">acephala</Emphasis> (DC.) Alef.) landraces,cultivars and wild populations in Europe

AFLP markers were used to characterize diversity and asses the genetic structure among 17 accessions of kale landraces, cultivars and wild populations from Europe. The range of average gene diversity in accessions was 0.11–0.27. Several landraces showed higher levels of diversity than the wild populations and one cultivar had the lowest diversity measures. The landraces that were most genetically diverse were from areas where kales are known to be extensively grown, suggesting in situ conservation in these areas as a supplement to storage of seeds in gene banks. An analysis of molecular variance (AMOVA) showed that 62% of the total variation was found within accessions. For most accessions, genetic distance was not related to geographic distance. Similarities among accessions were probably not caused by recent gene flow since they were widely separated geographically; more likely the relationship among them is due to seed dispersal through human interactions. Our results indicate that a kale population found in a natural habitat in Denmark was probably not truly wild but most likely an escape from a cultivated Danish kale that had subsequently become naturalized.     

Cytological and RAPD data revealed genetic relationships among nine selected populations of the wild bramble species, <Emphasis Type="Italic">Rubus</Emphasis><Emphasis Type="Italic">parvifolius</Emphasis> and <Emphasis Type="Italic">R. coreanus</Emphasis> (Rosaceae)

Rubus parvifolius L. and R. coreanus Miq. are the two morphologically distinct, endemic wild brambles in East Asia that may be useful in raspberry breeding and cultivar development. In this study, genetic relationships among a total of nine populations of these two species, collected from Sichuan Province in southwestern China, were investigated by cytological and RAPD analyzing. With the exception of ploidy levels, R. parvifolius and R. coreanus had similar cytological features. Six R. parvifolius populations included diploids, triploid, tetraploid, and mixed diploid-tetraploid cytotype plants; among them, the population that produced the fewest seeds was the triploid, while the three R. coreanus populations were all diploid. But obvious genetic differentiation between R. parvifolius and R. coreanus was revealed by RAPD markers, the Euclidean’s GS coefficient generated by UPGMA cluster analysis between R. parvifolius and R. coreanus was only 0.442, about half of that observed within species. Both genetic relationships among these populations and the origin of polyploidy in R. parvifolius populations are also discussed.     

Comparative Study of Common Bean (<Emphasis Type="Italic"> Phaseolus vulgaris</Emphasis>L.) Landraces Conserved <Emphasis Type="Italic">ex situ</Emphasis> in Genebanks and <Emphasis Type="Italic">in situ</Emphasis> by Farmers

Genetic diversity of populations stored ex situ or in situ can be altered due to the management practices they are subjected to. In this paper, we compare populations of two common bean (Phaseolus vulgaris L.) landraces grown on farms with material collected from the same farms and now kept in two ex situ collections (CIAT and REGEN) with the purpose to monitor any changes that have occurred due to ex situ conservation. The diversity was measured using seven bean microsatellite markers. Further phenotypic and developmental traits were registered in a field experiment. Compared with the in situ populations, the ex situ ones had a lower level of gene diversity and we suggest that this is due to the regeneration process. Most of the phenotypic traits did not differ significantly between ex situ and in situ populations, although for yield and 100-seed weight, the CIAT material showed significant lower values. We assume that these populations have gone through an adaptational change. Overall, the conservation ex situ has been successful in maintaining the majority of the adaptations found in the landraces studied, however, the probable loss of genetic diversity that we have observed, suggest that protocols for the regeneration process must be carefully worked out if the majority of alleles are to be preserved for the future. This study also highlights the complementarity of ex situ and in situ conservation methods in order to preserve landrace adaptations and to capture new, useful diversity generated in in situ populations.     

A significant proportion of indigenous rhizobia from India associated with soybean (<Emphasis Type="Italic">Glycine max</Emphasis> L.) distinctly belong to <Emphasis Type="Italic">Bradyrhizobium</Emphasis> and <Emphasis Type="Italic">Ensifer</Emphasis> genera

The diversity among 269 rhizobia isolated from naturally occurring root nodules of soybean collected from two different agro-ecological regions of India, based on RFLP and sequences of the intergenic spacer (IGS) between the 16S and 23S rRNA genes, growth rate, and indole acetic acid production, revealed their significant, site-dependent genomic diversity. Among these bacteria, nine IGS genotypes were identified with two endonucleases. They were distributed into five divergent lineages by sequence analysis of each IGS representative strain, i.e., (1) comprising IGS genotypes I, II, III, and reference Bradyrhizobium yuanmingense; (2) with genotype IV and strains of unclassified bradyrhizobia genomic species; (3) including genotypes V, VI, and Bradyrhizobium liaoningense; (4) with IGS genotype VII and Bradyrhizobium elkanii strains; and (5) comprising IGS genotypes VIII, IX, and different Ensifer genus bacteria. Host-specificity test revealed that all rhizobia-nodulated soybean and cowpea and only part of them formed nodules on Arachis hypogeae and Cajanus cajan. The great diversity of soybean nodulators observed in this study emphasises that Indian soil is an important reservoir of nitrogen-fixing rhizobia.     

Investigation of recent population bottlenecks in Kenyan wild sorghum populations (<Emphasis Type="Italic">Sorghum bicolor</Emphasis> (L.) Moench ssp. <Emphasis Type="Italic">verticilliflorum</Emphasis> (Steud.) De Wet) based on microsatellite diversity and genetic disequilibria

Identifying populations that have recently suffered a severe reduction in size is particularly important for their conservation as they are likely to suffer an increased risk of genetic erosion. We investigated the presence of recent bottlenecks in two wild sorghum populations from different eco-geographical conditions in Kenya employing 18 microsatellite markers. Microsatellite analysis showed high allelic diversity in the two populations, with a mean of 4.11 and 6.94 alleles per locus in the North-West wild sorghum population (NWWSP) and the South-East wild sorghum population (SEWSP), respectively. The mean observed heterozygosity was 0.34 and 0.56 in NWWSP and SEWSP, respectively. A large long-term effective populations size for both populations was observed assuming either an infinite allele model or a stepwise mutation model. There was no apparent loss of genetic variability for either of the populations. Test of heterozygosity excess indicated that a recent bottleneck in the two populations is highly unlikely. Furthermore, analysis of the allele frequency distribution revealed an L-shaped distribution which would not have been observed in case a recent bottleneck had reduced genetic variability in the two populations. The fact that most loci displayed a significant heterozygosity deficiency could be explained by population subdivision and the mixed mating system exhibited by wild sorghum populations. Furthermore, the possibility of a historical expansion of wild sorghum populations and presence of null alleles could not be ruled out.     

AFLP-based genetic diversity assessment among Chinese vegetable mustards (<Emphasis Type="Italic">Brassica juncea</Emphasis> (L.) Czern.)

Amplified fragment length polymorphism (AFLP) analysis was used to evaluate the genetic relationships and diversities of Chinese vegetable mustards. Fourteen pairs of primers generated a total of 366 scorable fragments among 16 accessions of Brassica juncea studied, of which 296 bands were polymorphic with an average of 21.1% polymorphic bands per primer combination. Genetic similarities were obtained using Nei and Li similarity coefficients, and a dendrogram of the 16 accessions was made by UPGMA clustering method. The Nei and Li Similarity coefficient value ranged from 0.63 to 0.88. This result indicated that the 16 accessions of B. juncea possessed high level genetic variations. The cluster analysis showed that the vegetable mustards could be grouped into two main groups and some minor rami, which was partially in accordance with the traditional classification that based on different edible organs of vegetable mustards. The incongruity between morphological and molecular classification might be attributed to the high selection pressure during domestication of Chinese vegetable mustards, producing some accessions with similar genetic backgrounds evolving into abundant morphological variations. The great diversification among Chinese vegetable mustards not only provides an excellent object for molecular evolution research of B. juncea but also is of great value for widening the genetic basis of breeding programs and breeding materials selection. Besides, our study also indicates that AFLP are informative and can provide significant insights for genetic diversity research in B. juncea.     

Diversity in cowpea (<Emphasis Type="Italic">Vigna unguiculata</Emphasis> (L.) Walp.) local populations from Greece

Cowpea cultivation in many countries around the Mediterranean Basin depends on a number of locally adapted populations conserved on-farm at a small scale, rather than on the use of modern varieties. Documentation, characterization and exploitation of traditional local populations could contribute to their conservation and utilization as sources of desirable characteristics. Therefore, a study was conducted to (a) characterize, (b) assess diversity and (c) classify 23 on-farm conserved local cowpea populations based on 32 agro-morphological traits. Investigations on diversity of characteristics related to seed yield, mineral and seed crude protein content as well as on correlations among them were carried out. A relatively high phenotypic diversity was observed. In particular, a high level of within population diversity was found (\( \bar{H}s \) = 0.34) exceeding that among populations’ diversity (Gst = 0.27). Principal component analysis classified the majority of local populations into two groups (mainly according to populations’ seed coat color and eye color), further divided into six subgroups regardless of the populations’ geographical origin. Significant differences were also observed among the populations studied for potassium and calcium, as well as for their seed crude protein content which ranged from 22.14 to 28.37 %. The results show appreciable levels of intra- and inter-phenotypic diversity in on-farm conserved cowpea populations, which indicates the existence of a valuable gene pool for future exploitation in breeding programs.     

<Emphasis Type="Italic">Achromobacter insolitus</Emphasis> and <Emphasis Type="Italic">Zoogloea ramigera</Emphasis> associated with wheat plants (<Emphasis Type="Italic">Triticum aestivum</Emphasis>)

This study reports for the first time the presence of diazotrophic bacteria belonging to the genera Achromobacter and Zoogloea associated with wheat plants. These bacterial strains were identified by the analysis of 16S rDNA sequences. The bacterium IAC-AT-8 was identified as Azospirillum brasiliense, whereas isolates IAC-HT-11 and IAC-HT-12 were identified as Achromobacter insolitus and Zoogloea ramigera, respectively. A greenhouse experiment involving a non-sterilized soil was carried out with the aim to study the endophytic feature of these strains. After 40 days from inoculation, all the strains were in the inner of roots, but they were not detected in soil. In order to assess the location inside wheat plants, an experiment was conducted under axenic conditions. Fifteen days after inoculation, preparations of inoculated plants were observed by the scanning electron microscope, using the cryofracture technique, and by the transmission electron microscope. It was observed that all isolates were present on the external part of the roots and in the inner part at the elongation region, in cortex cells, but not in the endodermis or in the vascular bundle region. No colonizing bacterial cells were observed in wheat leaves.     

Putative Role of <Emphasis Type="Italic">Flavobacterium</Emphasis>, <Emphasis Type="Italic">Dokdonella</Emphasis> and <Emphasis Type="Italic">Methylophilus</Emphasis> Strains in Paracetamol Biodegradation

Paracetamol, the most widely and globally used analgesic and antipyretic, is easily accumulated in aquatic environments. In the present study, the biodegradation of paracetamol in different media (one for general growth, one specific for sulfate reducing bacteria, a mineral salts medium and municipal wastewater) inoculated with two types of sludge (from anaerobic lagoon and from oxidation ditch) under different oxygenic conditions (anoxic; moderate oxygenation in open flasks and high oxygenation by aeration) was investigated. In addition, bacteria with relative abundances increasing simultaneously with paracetamol degradation, when this drug was the only carbon source, thus with a putative role in its degradation, were identified using 16S rRNA gene sequences. The results show that aerobic microorganisms had a major role in the degradation of paracetamol, with 50 mg/L totally removed from municipal wastewater after 2 days incubation with aeration, and that the metabolites 4-aminophenol and hydroquinone plus one compound not identified in this work were produced in the process. The identification of bacteria with a role in the degradation of paracetamol revealed a strain from genus Pseudomonas with the highest final relative abundance of 21.2%, confirming previous works reporting strains of this genus as paracetamol decomposers. Besides, genera Flavobacterium, Dokdonella and Methylophilus were also in evidence, with initial relative abundances of 1.66%, 1.48 and 0.00% (not detected) in the inoculum and 6.91%, 3.80 and 3.83% after incubation, respectively. Therefore, a putative role of these genera in paracetamol biodegradation is suggested for the first time.

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