Build-up of carbon fractions in technosol-biochar amended partially reclaimed mine soil grown with <Emphasis Type="Italic">Brassica juncea</Emphasis>

Purpose

Soil organic carbon (SOC) and its labile fractions are strong determinants of physical, chemical and biological properties. The objective of the present work was to evaluate the effects of organic amendments (technosol made of wastes and biochar) and Brassica juncea L. on the soil C fractions in a reclaimed mine soil.

Materials and methods

The studied soil was from a former copper mine that was subsequently partially reclaimed with vegetation and wastes. A greenhouse experiment was carried out to amend the mine soil with different proportions of technosol and biochar mixture and planting B. juncea. B. juncea plants can tolerate high levels of metals and can produce a large amount of biomass in relatively short periods of time.

Results and discussion

The results showed that with the addition of biochar and wastes, soil pH increased from 2.7 to 6.18, SOC from undetectable to 105 g kg^?1 and soil total nitrogen (TN) from undetectable to 11.4 g kg^?1. Amending with wastes and biochar also increased dissolved organic carbon (DOC) from undetectable to 5.82 g kg^?1, carbon in the free organic matter (FOM) from undetectable to 30.42 g kg^?1, FAP (carbon in fulvic acids removed with phosphoric acid) from undetectable to 24.14 g kg^?1 and also increased the humification ratio, the humification index, the polymerisation rate and the organic carbon in the humified fractions (humic acids, fulvic acids and humin). Soils amended and vegetated with B. juncea showed lower FOM values and higher humification index values than the soils amended only with biochar and wastes.

Conclusions

This study concludes that the combined addition of wastes and biochar has a greater potential for both increasing and improving organic carbon fractions in mine soils. The authors recommend the application of biochar and technosol made of wastes as a soil amendment combined with B. juncea on soils that are deficient in organic matter, since they increased all of the SOC fractions in the studied copper mine soil.

     

Eco-restoration of a mine technosol according to biochar particle size and dose application: study of soil physico-chemical properties and phytostabilization capacities of <Emphasis Type="Italic">Salix viminalis</Emphasis>

Purpose

Anthropic activities induce severe metal(loid)s contamination of many sites, which is a threat to the environment and to public health. Indeed metal(loid)s cannot be degraded, and thus accumulate in soils. Furthermore, they can contaminate surrounding ecosystems through run-off or wind erosion. This study aims to evaluate the phytostabilization capacity of Salix viminalis to remediate As and Pb highly contaminated mine site, in a biochar-assisted phytoremediation context and to assess biochar particle size and dose application effects.

Materials and methods

To achieve this, mesocosm experiments were conducted using the contaminated technosol and four different size fraction of one biochar as amendment, at two application rates (2 and 5%). Non-rooted cuttings of Salix viminalis were planted in the different mixtures. In order to characterize the mixtures, soil pore waters were sampled at the beginning and at the end of the experiment and analyzed for pH, electrical conductivity, and metal(loid) concentrations. After 46 days of Salix growth, roots, stems, and leaves were harvested and weighed, and As and Pb concentrations and distributions were measured.

Results and discussion

Soil fertility improved (acidity decrease, electrical conductivity increase) following biochar addition, whatever the particle size, and the Pb concentration in soil pore water decreased. Salix viminalis did not grow on the non-amended contaminated soil while the biochar amendment permitted its growth, with a better growth with the finest biochars. The metal(loid)s accumulated preferentially in roots.

Conclusions

Fine biochar particles allowed S. viminalis growth on the contaminated soil, allowing this species to be used for technosol phytostabilization.

     

Characterization of fertile amphidiploid between <Emphasis Type="Italic">Raphanus sativus</Emphasis> and <Emphasis Type="Italic">Brassica alboglabra</Emphasis> and the crossability with <Emphasis Type="Italic">Brassica</Emphasis> species

A fertile amphidiploid × Brassicoraphanus (RRCC, 2n = 36) between Raphanus sativus cv. HQ-04 (2n = 18, RR) and Brassica alboglabra Bailey (2n = 18, CC) was synthesized and successive selections for seed fertility were made from F₄ to F₁₀. F₁₀ plants exhibited good fertility with 14.9 seeds per siliqua and 32.3 g seeds per plant. Cytological observation revealed that frequent secondary pairing occurred among 3 chromosome pairs in pollen mother cells of plants (F₄) with lower fertility, but not of plants with high fertility (F₁₀). GISH analysis indicated that these F₁₀ plants included the expected 18 chromosomes from R. sativus and B. alboglabra, respectively, but they lost approximately 27.6% R. sativus and 35.6% B. alboglabra AFLP (amplified fragment length polymorphism) bands. The crossability of the Raphanobrassica with R. sativus and 5 Brassica species (13 cultivars) were investigated. Seeds or F₁ seedlings were easy to be produced from crosses × Brassicoraphanus × R. sativus, and B. napus, B. juncea and B. carinata × Brassicoraphanus. Fewer seeds or seedlings were obtained from crosses × Brassicoraphanus × B. napus, B. juncea and B. carinata. However, few seeds were harvested in the reciprocals of × Brassicoraphanus with B. rapa and B. oleracea. The possible cause of fertility improvements and the potential of the present × Brassicoraphanus for breeding were discussed.     

Phytotransformation of Phorate by <Emphasis Type="BoldItalic">Brassica juncea</Emphasis> (Indian Mustard)

Over 5 days, Brassica juncea removed 54% of the highly toxic insecticide phorate from the medium with the formation of phorate sulfoxide in small quantity. The loss of phorate from the medium followed first-order kinetics. The half-life of phorate disappearance from water decreased by ~4.5-fold in the presence of B. juncea. Mild phorate phytotoxicity was evident from the elevated activities of the antioxidative enzymes like glutathione-disulfide reductase, glutathione S-transferase, superoxide dismutase, and catalase in the plants. Nevertheless, the ubiquitous antioxidative peroxidase was not significantly increased, nor the total glutathione content, due to phorate exposure. Phosphotriester bond hydrolysis and glutathione S-transferase-mediated conjugation seemed to be the key reactions for phorate metabolism by B. juncea. From the limited information available, for the first time, a tentative mapping of phytotransformation pathways was performed.     

Genetic diversity in oil and vegetable mustard (<Emphasis Type="Italic">Brassica</Emphasis><Emphasis Type="Italic">juncea</Emphasis>) landraces revealed by SRAP markers

Mustard (Brassica juncea) is an important crop in both ancient and modern world. It has a broad resource of genetic diversity that is used primarily as oilseed but as vegetables, condiment and medicines also. Its superior tolerance to adverse environments, e.g., drought, high temperature and low fertility suggests its better adaptability in future possible harsh environments. Chinese vegetable mustard displays a wide spectrum of morphotypes. A collection of 95 accessions of B. juncea representing oil and vegetable mustards from China, France, India, Pakistan, and Japan were assessed to determine diversity at the molecular level using sequence-related amplified polymorphism (SRAP). Eight SRAP primer combinations identified a total of 326 scorable fragments of which 161 were polymorphic (49.39%). The percentage of polymorphism for each primer combination varied from 21.88 to 66.67%. Both Shannon-Weaver and Simpson genetic diversity index indicated that the level of genetic diversity within vegetable mustard is much higher than within oil mustard, and also winter oil mustards are genetically more diverse than spring oil mustards. Based on the Cluster and Principal Coordinates analysis, which were conducted on the similarity matrix of SRAP marker data, vegetable, spring oil and winter oil mustard were clearly divided into three distinct groups and among these three groups, spring and winter oil mustard are geneticlly closer than vegetable mustard. This suggests that bilateral gene exchange between oil and vegetable gene pools in the breeding program will effectively elevate the genetic potential in developing higher yields, more disease resistance, better quality and better adapted lines.     

Production and cytogenetics of trigeneric hybrid involving <Emphasis Type="Italic">Triticum</Emphasis>, <Emphasis Type="Italic">Psathyrostachys</Emphasis> and <Emphasis Type="Italic">Secale</Emphasis>

Trigeneric hybrids may help establish evolutionary relationships among different genomes present in the same cellular-genetic background, and also offers the possibility to transfer different alien characters into cultivated wheat. In this study, a new trigeneric hybrid involving species from the Triticum, Psathyrostachys and Secale was synthesized by crossing wheat-P. huashanica amphiploid (PHW-SA) with wheat-S. cereale amphiploid (Zhongsi 828). The crossability of F₁ hybrid was high with 35.13%, and the fertility was 41.95%. The morphological characteristics of F₁ plants resembled the parent Zhongsi 828. The trigeneric hybrids pollen mother cells (PMCs) regularly revealed averagely 19.88 univalents, 9.63 ring bivalents, 3.97 rod bivalents, 0.60 trivalents and 0.03 tetravalents per cell. Multivalents consisted of trivalents and tetravalents can be observed in 52.7% of cells. A variation of abnormal lagging chromosome, micronuclei and chromosome bridge were formed at anaphase I and telophase II. The mean chromosomes number of F₂ progenies was 2n = 46.13, and the distribution range was 42–53. GISH results revealed that most F₂ plants had 6–12 S. cereale chromosomes, and only 0–2 P. huashanica chromosomes were detected. The results indicated that S. cereale chromosomes can be preferentially transmitted in the F₂ progenies of trigeneric hybrid than P. huashanica chromosomes. A survey of disease resistances revealed that the stripe rust resistance from the PHW-SA were completely expressed in the F₁ and some F₂ plants. The trigeneric hybrid could be a useful bridge for the transference of P. huashanica and S. cereale chromatins to common wheat.     

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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Graphical Abstract ?

     

<Emphasis Type="Italic">Rhizobium</Emphasis>,<Emphasis Type="Italic"> Bradyrhizobium</Emphasis> and<Emphasis Type="Italic"> Agrobacterium</Emphasis> strains isolated from cultivated legumes

The present study was conducted to isolate and characterize rhizobial strains from root nodules of cultivated legumes, i.e. chickpea, mungbean, pea and siratro. Preliminary characterization of these isolates was done on the basis of plant infectivity test, acetylene reduction assay, C-source utilization, phosphate solubilization, phytohormones and polysaccharide production. The plant infectivity test and acetylene reduction assay showed effective root nodule formation by all the isolates on their respective hosts, except for chickpea isolate Ca-18 that failed to infect its original host. All strains showed homology to a typical Rhizobium strain on the basis of growth pattern, C-source utilization and polysaccharide production. The strain Ca-18 was characterized by its phosphate solubilization and indole acetic acid (IAA) production. The genetic relationship of the six rhizobial strains was carried out by random amplified polymorphic DNA (RAPD) including a reference strain of Bradyrhizobium japonicum TAL-102. Analysis conducted with 60 primers discriminated between the strains of Rhizobium and Bradyrhizobium in two different clusters. One of the primers, OPB-5, yielded a unique RAPD pattern for the six strains and well discriminated the non-nodulating chickpea isolate Ca-18 from all the other nodulating rhizobial strains. Isolate Ca-18 showed the least homology of 15% and 18% with Rhizobium and Bradyrhizobium, respectively, and was probably not a (Brady)rhizobium strain. Partial 16S rRNA gene sequence analysis for MN-S, TAL-102 and Ca-18 strains showed 97% homology between MN-S and TAL-102 strains, supporting the view that they were strains of B. japonicum species. The non-infective isolate Ca-18 was 67% different from the other two strains and probably was an Agrobacterium strain.     

ISSR variation within and among wild <Emphasis Type="Italic">Brassica juncea</Emphasis> populations: implication for herbicide resistance evolution

There is a need to understand whether weed genetic diversity is the same among different populations, especially between those exposed to herbicide selection and other without exposure history. Inter-simple sequence repeat (ISSR) were used to assess level and patterns of genetic diversity in wild Brassica juncea (L.) Czern. et Coss. populations. A total of 93 plants from 24 wild populations in China were analysed by eight primers resulting in 86 highly reproducible ISSR bands. The analysis of molecular variance (AMOVA) with distances among individuals corrected for the dominant nature of ISSRs showed that most of the variation (54.09%) occurred among populations, and the remaining 45.91% variance was attributed to differences among individuals within populations. The high differentiation was, perhaps, due to limited gene flow (Nm < 1.0) of this species. Though highest gene diversity was observed in resistant B. juncea population, the overall distribution of diversity across China was not geographic dependent. High F _ST value (0.541) corroborated AMOVA partitioning and provided significant evidence for population differentiation in wild B. juncea. UPGMA cluster analyses, based on Nei’s genetic distance, revealed grouping pattern geographically. Based on these results, the factors affect weed population genetic diversity and implication for herbicide resistance evolution were discussed in the context of transgenic crops advent and increasing herbicide usage in China.     

Characterization of the gibberellic acid response of the <Emphasis Type="Italic">Brassica napus</Emphasis> L. em. Metzg. dwarf mutant <Emphasis Type="Italic">NDF</Emphasis>-<Emphasis Type="Italic">1</Emphasis>

A novel dwarf mutant of Brassica napus L. em. Metzg., named NDF-1, was derived from a high doubled haploid line ‘3529’ of which seeds were jointly treated with chemical inducers and fast neutron bombardment. The germination results showed that the germination of NDF-1 was insensitive in response to exogenous gibberellic acid 3 (GA₃). The studies on growth response to exogenous GA₃ showed that NDF-1 seeding has at least 10-fold insensitivity than the wild-type. Moreover, no matter what concentrations of GA₃ were added to the seedlings and adult plants, the NDF-1 could not restore the wild type phenotype. These results indicated that the B. napus dwarf mutant NDF-1 was GA-insensitive mutant. The histological observations showed that the key reason of leading NDF-1 to dwarf was the reduction of hypocotyls and stems cell numbers.     

<Emphasis Type="Italic">Trichoderma</Emphasis> improves the growth of <Emphasis Type="Italic">Leymus chinensis</Emphasis>

Bio-fertilizer application has been proposed as a strategy for enhancing soil fertility, regulating soil microflora composition, and improving crop yields, and it has been widely applied in the agricultural yields. However, the application of bio-fertilizer in grassland has been poorly studied. We conducted in situ and pot experiments to investigate the practical effects of different fertilization regimes on Leymus chinensis growth, with a focus on the potential microecological mechanisms underlying the responses of soil microbial composition. L. chinensis biomass was significantly (P?<?0.05) increased by treatment with 6000 kg ha^?1 of Trichoderma bio-fertilizer compared with other treatments. We found a positive (R² =?0.6274, P <?0.001) correlation between bacterial alpha diversity and L. chinensis biomass. Hierarchical cluster analysis and nonmetric multidimensional scaling (NMDS) revealed that soil bacterial and fungal community compositions were all separated according to the fertilization regime used. The relative abundance of the most beneficial genera in bio-fertilizer (BOF) (6000 kg ha^?1Trichoderma bio-fertilizer) was significantly higher than in organic fertilizer (OF) (6000 kg ha^?1 organic fertilizer) or in CK (non-amend fertilizer), there the potential pathogenic genera were reduced. There were significant negative (P?<?0.05) correlations between L. chinensis biomass and the relative abundance of several potential pathogenic genera. However, the relative abundance of most beneficial genera were significantly (P?<?0.05) positively correlated with L. chinensis biomass. Soil properties had different effects on these beneficial and on these pathogenic genera, further influencing L. chinensis biomass.     

<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.     

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.     

Polymorphism of <Emphasis Type="Italic">Got2</Emphasis> DNA sequences sheds light on <Emphasis Type="Italic">Aegilops tauschii</Emphasis> Coss. intraspecies divergence and origin of <Emphasis Type="Italic">Triticum aestivum</Emphasis> L.

DNA sequences of nuclear gene Got2 was studied in 60 accessions of Aegilops tauschii, 29 of subsp. tauschii and 31 of subsp. strangulata. It was found that Got2 allozyme polymorphism in Ae. tauschii is due to a single, unique, mutation which led to replacement of glutamic acid by isoleucine in residue 256 of the enzyme molecule, encoded by Got2. As revealed by Got2 DNA sequences variation, initially in its history Ae. tauschii was presented by subsp. strangulata, and among phylogenetic lineages of subsp. strangulata, the lineage “t-9¹s” (TauL3) is the most ancient, a relict one. Subspecies tauschii is relatively “young”. Initially it was presented by the lineage marked by combination of allozyme alleles Got2 ¹⁰⁵ and Acph1 ¹⁰⁰. In the past it inhabited the Continental area from Caucasia to Pakistan, but later on it was forced out by newly originated, now—a major lineage of subsp. tauschii, marked by Got2 ¹⁰⁰. This lineage extended the Continental area of the species up to Kirgizstan, but actually failed to penetrate into pre-Caspian area, occupied by subsp. strangulata. These results essentially differ from those obtained previously, using chloroplast DNA (cpDNA) sequences polymorphism. As revealed by cpDNA, the major, “usual”, subsp. strangulata (TauL2) is “younger” than subsp. tauschii, which resided on phylogenetic tree between relict lineage “t-9¹s”of subsp. strangulata—and major subsp. strangulata. But both cpDNA and Got2 DNA sequences indicate that the level of genetic variation in subsp. tauschii is much lower than in subsp. strangulata. According to Got2 DNA sequences variation, it was Ae. tauschii subsp. strangulata lineage “k-109″ which donated genome D to Triticum aestivum L. This lineage includes accessions: k-109 from South-Eastern Precaspian Azerbaijan; KU-2105, KU-2159 from Western Precaspian Iran; KU-2080 from Eastern Precaspian Iran.     

A new mycorrhizal helper bacterium, <Emphasis Type="Italic">Ralstonia</Emphasis> species,in the ectomycorrhizal symbiosis between <Emphasis Type="Italic">Pinus thunbergii</Emphasis> and <Emphasis Type="Italic">Suillus granulatus</Emphasis>

In a Robinia-pseudoacacia-dominated coastal forest in Tottori prefecture Japan, the growth and survival of Pinus thunbergii seedlings and the natural regeneration of P. thunbergii was disturbed by R. pseudoacacia. In order to improve the growth of P. thunbergii seedling in the Tottori sand dune, we tried to find a mycorrhiza helper bacteria (MHB) from P. thunbergii mycorrhizosphere in a Tottori sand dune. Two MHB, Ralstonia sp. and Bacillus subtilis, were selected from the nine bacterial species isolated from the mycorrhizosphere of P. thunbergii. The bacterial effect on the ectomycorrhizal fungus Suillus granulatus was investigated by confrontation assay and a microcosm experiment. The confrontation assay showed that Ralstonia sp. promoted the hyphal growth of S. granulatus. Moreover, the S. granulatus–P. thunbergii symbiosis was significantly stimulated by Ralstonia sp. and B. subtilis. Ralstonia sp. and B. subtilis were regarded as MHB associated with P. thunbergii. This is the first report of Ralstonia sp. as an MHB.     

Salt tolerance in <Emphasis Type="Italic">Zea mays</Emphasis> (L). following inoculation with <Emphasis Type="Italic">Rhizobium</Emphasis> and <Emphasis Type="Italic">Pseudomonas</Emphasis>

This study aimed to investigate the effect of inoculation with plant growth-promoting Rhizobium and Pseudomonas species on NaCl-affected maize. Two cultivars of maize (cv. Agaiti 2002 and cv. Av 4001) selected on the basis of their yield potential were grown in pots outdoors under natural conditions during July. Microorganisms were applied at seedling stage and salt stress was induced 21 days after sowing and maintained up to 50% flowering after 120 days of stress. The salt treatment caused a detrimental effect on growth and development of plants. Co-inoculation resulted in some positive adaptative responses of maize plants under salinity. The salt tolerance from inoculation was generally mediated by decreases in electrolyte leakage and in osmotic potential, an increase in osmoregulant (proline) production, maintenance of relative water content of leaves, and selective uptake of K ions. Generally, the microbial strain acted synergistically. However, under unstressed conditions, Rhizobium was more effective than Pseudomonas but under salt stress the favorable effect was observed even if some exceptions were also observed. The maize cv. Agaiti 2002 appeared to be more responsive to inoculation and was relatively less tolerant to salt compared to that of cv. Av 4001.     

Genetic Characterization of Brazilian Annual <Emphasis Type="Italic">Arachis</Emphasis> Species from Sections <Emphasis Type="Italic">Arachis</Emphasis> and <Emphasis Type="Italic">Heteranthae</Emphasis> using RAPD Markers

The genus Arachis is divided into nine taxonomic sections. Section Arachis is composed of annual and perennial species, while section Heteranthae has only annual species. The objective of this study was to investigate the genetic relationships among 15 Brazilian annual accessions from Arachis and Heteranthae using RAPD markers. Twenty-seven primers were tested, of which nine produced unique fingerprintings for all the accessions studied. A total of 88 polymorphic fragments were scored and the number of fragments per primer varied from 6 to 17 with a mean of 9.8. Two specific markers were identified for species with 2n = 18 chromosomes. The phenogram derived from the RAPD data corroborated the morphological classification. The bootstrap analysis divided the genotypes into two significant clusters. The first cluster contained all the section Arachis species, and the accessions within it were grouped based upon the presence or absence of the ‘A’ pair and the number of chromosomes. The second cluster grouped all accessions belonging to section Heteranthae.     

Genetic diversity,genetic structure and migration routes of wild <Emphasis Type="Italic">Brassica juncea</Emphasis> in China assessed by SSR markers

The wild Brassica juncea (L.) Czern. et Coss., systematically belonging to the genus Brassica L. in the Cruciferae family, has become a noxious weed for cropping systems nowadays. Here, simple sequence repeat (SSR) markers were applied to investigate the genetic diversity, genetic structure and migration routes of the wild B. juncea populations in China. The results showed that a total of 90 alleles, with extensive allelic diversity, were observed at the 11 SSR loci of the wild B. juncea. The STRUCTURE analysis indicated that all the 25 wild populations were best described as belonging to two lineages. High Fst value (0.568), together with the partitioning, provided significant evidence for lineage differentiation in wild B. juncea. The high differentiation between the two lineages was, perhaps, due to limited gene flow (Nm?=?0.301) of this species. The analysis of molecular variance with distances among individuals corrected for the dominant nature of SSRs showed that most of the variation (59%) occurred within populations, and the remaining 41% variance was attributed to differences among populations. The distribution of diversity across China was significantly geographically dependent. NJ cluster analysis, based on genetic distance, grouped populations geographically, which further corroborated spatial pattern of two lineages. Based on these results, two routes were proposed for the migration of wild B. juncea in China after its origin from northwest China, heading east along the Yellow River or Yangtze River, respectively. We concluded that China, especially the northwest, is one of the primary origins of B. juncea.     

Phylogenetic relationships between <Emphasis Type="Italic">Ensete</Emphasis> and <Emphasis Type="Italic">Musa</Emphasis> species as revealed by the <Emphasis Type="Italic">trnT trnF</Emphasis> region of cpDNA

Complete sequences of transcribed spacers and introns from the trnT trnF region of chloroplast DNA (cp DNA) were generated from Musaceae species to establish the phylogenetic relationships among 3 species of Ensete including the economically important Ensete ventricosum (Welw.) Cheesman and 13 species of Musa. Parsimony analysis and pair wise distance data produced a single tree, with Ensete and Musa as clearly distinguished clades. Six Musa and three Ensete clades were generated. The topology of these clades did not change when the data were split into spacers and introns, although the split resulted in poor bootstrap support. Removing a hotspot from the entire data set improved clade support. The clades produced are discussed with reference to existing taxonomic and phylogenetic treatments. In contrast to previous suggestions, most of the Rhodochlamys species that we investigated clustered together with strong support establishing their distinctiveness from the Musa species studied. Ensete glaucum (Roxb.) Cheesman and Musa beccartii Simmonds appear to represent ancestral forms of Ensete and Musa, respectively for the presently studied species, and both genera have a common ancestor that is yet to be established. Our data also show that E. ventricosum cannot be reduced to E. glaucum, nor can E. gilleti (De Wild.) Cheesman be reduced to E. ventricosum, as some authorities have suggested. Ensete gilleti or a species very close to it appears to be the ancestral species of E. ventricosum.     

Morphological variation in <Emphasis Type="Italic">Laelia anceps</Emphasis> subsp. <Emphasis Type="Italic">dawsonii</Emphasis> f. <Emphasis Type="Italic">chilapensis</Emphasis> Soto-Arenas Orchidaceae in traditional home gardens of Chilapa,Guerrero, Mexico

Calaverita an endangered Mexican orchid is one of the most elegant and showy orchids of the genus Laelia and essential symbol within the local traditional celebrations of the Day of the Dead, in the region of Chilapa-Guerrero. We investigated morphological variation in flowers of la calaverita (Laelia anceps Lind. subsp. dawsonii (J. Anderson) Rolfe f. chilapensis Soto-Arenas, Orchidaceae) using the labellum of the specimens cultivated in traditional home gardens in the region of Chilapa, Guerrero, Mexico. The use of morphometric traits of the labellum is useful in appraising infra-subspecific morphological variation within the chilapensis form. Floral morphological variation exists among the specimens examined. These specimens, found exclusively in traditional home gardens, were grouped into four morphotypes: NAT, Topiltepec, Xulchuchío, and Terrero. The study of variation of la calaverita revealed the existence of four local morphotypes that originated from a long and complex process of selection of traits which responds to traditional preferences and knowledge about specific biotic and agroecological conditions. The cultural and aesthetic influence of traditional homegarden owners in the Chilapa region acts directly on selection, use and conservation of the morphological traits of la calaverita.