Towards an integrated linkage map of common bean

Summary Two genomic libraries were established to provide markers to develop an integrated map combining molecular markers and genes for qualitative and quantitative morpho-agronomic traits in common bean. Contrasting characteristics were observed for the two libraries. While 89% of the PstI clones were classified as single-copy sequences, only 21% of the EcoRIBamHI clones belonged in that category. Clones of these two libraries were hybridized against genomic DNA of nine genotypes chosen according to their divergent evolutionary origin and contrasting agronomic traits. Eight restriction enzymes were used in this study. PstI clones revealed 80–90% polymorphism between the Andean and Middle American gene pools and 50–60% polymorphism within these gene pools. However, under the same conditions only 30% of the EcoRI-BamHI clones showed polymorphism between the Middle American and Andean gene pools. Hybridization with PstI clones to EcoRI-, EcoRV-, or HindIII-digested genomic DNA resulted in a cumulative frequency of polymorphism of approximately 80%. Hybridizations to BamHI-, HaeIII-, HinfI-, PstI-, and XbaI-digested genomic DNA detected no additional polymorphisms not revealed by the former three enzymes. In the PstI library, a positive correlation was observed between the average size of hybridizing restriction fragments and the frequency of polymorphism detected by each restriction enzyme. This relationship is consistent with the higher proportion of insertion/deletion events compared with the frequency of nucleotide substitutions observed in that library.     

Restriction fragment length polymorphisms of the phytohemagglutinin genes inPhaseolus andVigna (Leguminosae)

Restriction fragment length polymorphisms at the phytohemagglutinin (PHA) locus were determined among 21 genotypes ofPhaseolus vulgaris, P. coccineus, P. acutifolius, P. lunatus, and threeVigna species, using five restriction enzymes and one double digestion, in order to provide molecular evidence for their genetic relatedness. The dissimilarity between genotypes was estimated from binary RFLP data. The dissimilarity was high among species (from 0.75 to 0.95), and of variable extent among genotypes of the same species (0.33–0.89). InP. vulgaris, two different DNA hybridization patterns were found, giving further evidence for two major gene pools in that species. The restriction patterns ofP. vulgaris var.aborigineus, the putative ancestral form ofP. vulgaris, exhibit clear homology toP. vulgaris genotypes. An undefined landrace from Taiwan could be identified as aP. vulgaris genotype. RFLP-based trees for the phytohemagglutinin genes of the species studied were computed with several distance matrix and parsimony methods.     

Detection of interspecific and intraspecific variation in Panicum millets through random amplified polymorphic DNA

The potential use of random amplified polymorphic DNA (RAPD) was evaluated as a source of genetic markers for studying variation among four species of Panicum and within the crop species P. miliaceum and P. sumatrense. Polymorphism in RAPD markers was observed across and within species. The four species were distinct in RAPD patterns and were separated at low correlation values even with small samples involving single genotypes per species. Accessions of P. miliaceum were grouped according to geographical regions of origin. The study demonstrated that unlike isozyme and protein electrophoresis patterns, RAPD markers can be applied to studying genetic diversity, defining gene pools, and identifying cultivars for this group of millets.     

Elicitation studies in cell suspension cultures of Cannabis sativa L.

Cannabis sativa L. plants produce a diverse array of secondary metabolites. Cannabis cell cultures were treated with biotic and abiotic elicitors to evaluate their effect on secondary metabolism. Metabolic profiles analysed by ¹H NMR spectroscopy and principal component analysis (PCA) showed variations in some of the metabolite pools. However, no cannabinoids were found in either control or elicited cannabis cell cultures. Tetrahydrocannabinolic acid (THCA) synthase gene expression was monitored during a time course. Results suggest that other components in the signaling pathway can be controlling the cannabinoid pathway.     

Induced defences in an endangered amphibian in response to an introduced snake predator

Introduced species have contributed significantly to the extinction of endemic species on islands. They also create new selection pressures on their prey that may result in modified life history strategies. Introduced viperine snakes (Natrix maura) have been implicated in the decline of the endemic midwife toad of Mallorca (Alytes muletensis). A comparison of A. muletensis tadpoles in natural pools with and without snakes showed that those populations subject to snake predation possessed longer tails with narrower tail fins but deeper tail muscles. Field and laboratory experiments showed that these changes in tail morphology could be induced by chemical and tactile cues from snakes. Populations of tadpoles that were subject to snake predation also displayed clear bimodal size-frequency distributions, with intermediate-sized tadpoles missing from the pools completely. Tadpoles in pools frequented by snakes developed faster in relation to their body size than those in pools without snakes. Variation in morphology between toad populations may therefore be caused by a combination of size-selective predation and tadpole plasticity. The results of this study indicate that the introduction of alien species can result in selection for induced defences, which may facilitate coexistence between predator and prey under certain conditions.     

Isozyme polymorphism and organization of the agamic complex of the Maximae (Panicum maximum Jacq., P. infestum Anders,and P. trichocladum K. Schum.) in Tanzania

The tribe Maximae (Panicum maximum Jacq., P. infestum Anders., P. trichocladum K. Schum.) includes two sympatric pools with different modes of reproduction and ploidy levels: an apomictic and tetraploid pool on the one hand, and a smaller, sexual and diploid pool on the other. From an analysis of isozyme polymorphism five main results were evident. First, overall polymorphism is considerable showing that apomixis does not lead to a reduction in diversity. Second, the isozyme polymorphism of the two pools is similar, and this may be explained by reciprocal gene flow (low, but continuous) between these two pools. Third, maximum local polymorphism is due to the simultaneous presence of P. maximum sexuals and P. infestum apomicts. A continuum exists between the two species. Fourth, a high proportion of rare alleles, arising from introgression from P. infestum, characterized the isozyme polymorphism. These rare alleles, strongly counter-selected at the diploid level, are maintained by apomixis; the frequency of triplex or quadruplex genotypes was nevertheless low. Fifth, the heterozygosity level within apomicts is not higher than that of sexuals, showing that the apomixis-polyploidy combination does not lead to a higher frequency of very heterozygous individuals.     

Transformation of Brassica juncea (L.) Czern with bacterial codA gene enhances its tolerance to salt stress

The codA gene for biosynthesis of glycinebetaine from Arthrobacter globiformis was used for transforming Brassica juncea cv. Pusa Jaikisan (which lack any means to synthesize glycinebetaine) through Agrobacterium mediated transformation. The stable insertion of the codA gene in the shoots obtained on medium with kanamycin and hygromycin was confirmed by PCR analysis of the nptII gene. Southern hybridization with a codA probe further demonstrated its successful integration. Immunoblot analysis revealed the presence of choline oxidase demonstrating that the bacterial codA gene had been successfully transcribed and translated. The seeds of transgenic lines showed enhanced capacity to germinate under salt stress as compared to that of the wild type. Further, the seedlings of transgenic plants that expressed codA gene showed significantly higher growth than that of the wild type under salt stress conditions. These results demonstrated that the introduction of a biosynthetic pathway for glycinebetaine into Brassica juncea significantly enhanced their salt tolerance. Hence, homozygous genotypes of selected transformed lines can be exploited for improving the salt tolerance of the desirable cultivars of Brassica juncea through breeding programmes.     

Prevalence of the prothrombin G20210A polymorphism in the Lebanese population: use of a reverse hybridization strip assay approach

The factor II (prothrombin) G20210A gene polymorphism is the second most common SNP reported in VTE where it is associated with elevated plasma prothrombin levels and with a 3-fold increased risk. We studied the distribution of the G/G, G/A, and A/A genotypes of the Prothrombin G20210A gene mutation in the general Lebanese population using a novel technique in order to assess their prevalence, compare the results to previously reported data and to describe an available method that will permit easy and fast identification of the mutation. Prothrombin different genotypes were determined using the Cardiovascular Disease (CVD) StripAssay which is based on a Polymerase Chain Reaction-Reverse hybridization technique and DNA from 205 unrelated healthy donors from our HLA-bank was used. The prevalence of G/G, G/A, and A/A genotypes was found to be 98.54, 1.46, and 0%, respectively, with G and A allelic frequency of 99 and 1%, respectively. The sampled Lebanese population showed prothrombin genotypes distribution similar to Caucasians, and our results are comparable to other reports on the Lebanese healthy individuals. However, this is the first report on the prevalence of prothrombin G20210A mutation using this technique. Our results suggest that this approach is reliable and can be used as an assessment for thrombophilia profile. In addition, future investigations should be conducted to assess the contribution of the prothrombin G20210A mutation, on its own and in collaboration with other factors, in various clinical entities notably VTE.     

High altitude mountain streams as a possible refuge habitat for the catfish <Emphasis Type="Italic">Amphilius uranoscopus</Emphasis>

Amphilius uranoscopus is a catfish species, restricted to rivers and streams in east, southern and central Africa. It is likely to be displaced due to both competition and predation by exotic trout and other introduced fish. In high altitude mountain streams it can be the only species occurring, which means that this habitat may act as a refuge for this species. Ecosystems like this are threatened by habitat alteration and are therefore in need of protection. The abiotic environment, population structure, behavior and feeding biology of Amphilius uranoscopus were studied in a small, high-altitude perennial tributary of the Limpopo River in the Soutpansberg mountain range, Limpopo Province, South Africa, during 2005–2006. Here A. uranoscopus showed nocturnal behavior. It used dark hollow crevices in rapids as shelters during the daytime. The rapids are characterized by a high flow rate, high dissolved oxygen content and coarse riverbed substrate consisting mainly of boulders without fallen leaves. In contrast to the adults, juveniles found shelter among the fallen leaves in pools. At night, A. uranoscopus moved out of the rapids into the open water of the pools. The main food of A. uranoscopus consisted of macroinvertebrates, mainly Trichoptera larvae. Amounts of algae and detritus in its diet were negligible. A. uranoscopus foraged mainly in rapids and on rock surfaces, spending less time foraging between the fallen leaves at the bottom of pools, in open water, at the surfaces of bottom and bank and near the water surface. At the water surface, it also fed opportunistically on terrestrial insects that dropped into the stream, like flying termites. A. uranoscopus was the top predator of the stream system and reached a high density (0.71 fish m⁻²). There was no competition from other fish species at the study site as they were lacking. A. uranoscopus showed a striking ability to climb and cling on to vertical substrates to conquer waterfalls and very shallow streams that sometimes become dry, using its fins, adhesive body and protruding mouth teeth.     

Matching population diversity of rhizobial nodA and legume NFR5 genes in plant–microbe symbiosis

We hypothesized that population diversities of partners in nitrogen‐fixing rhizobium–legume symbiosis can be matched for “interplaying” genes. We tested this hypothesis using data on nucleotide polymorphism of symbiotic genes encoding two components of the plant–bacteria signaling system: (a) the rhizobial nodA acyltransferase involved in the fatty acid tail decoration of the Nod factor (signaling molecule); (b) the plant NFR5 receptor required for Nod factor binding. We collected three wild‐growing legume species together with soil samples adjacent to the roots from one large 25‐year fallow: Vicia sativa, Lathyrus pratensis, and Trifolium hybridum nodulated by one of the two Rhizobium leguminosarum biovars (viciae and trifolii). For each plant species, we prepared three pools for DNA extraction and further sequencing: the plant pool (30 plant indiv.), the nodule pool (90 nodules), and the soil pool (30 samples). We observed the following statistically significant conclusions: (a) a monotonic relationship between the diversity in the plant NFR5 gene pools and the nodule rhizobial nodA gene pools; (b) higher topological similarity of the NFR5 gene tree with the nodA gene tree of the nodule pool, than with the nodA gene tree of the soil pool. Both nonsynonymous diversity and Tajima's D were increased in the nodule pools compared with the soil pools, consistent with relaxation of negative selection and/or admixture of balancing selection. We propose that the observed genetic concordance between NFR5 gene pools and nodule nodA gene pools arises from the selection of particular genotypes of the nodA gene by the host plant.     

RAPD markers linked to the Vf gene for scab resistance in apple

Scab (Venturia inaequalis) is one of the most harmful diseases of apple, significantly affecting world apple production. The identification and early selection of resistant genotypes by molecular markers would greatly improve breeding strategies. Bulked segregant analysis was chosen for the identification of RAPD markers linked to the Vf scab resistant gene. Five different RAPD markers, derived from the wild species Malus floribunda. 821, were identified, and their genetic distance from Vf gene was estimated. The markers OPAM19₂₂₀₀ and OPAL07₅₈₀ were found to be very closely linked to the Vf gene. This result was indirectly confirmed by the analysis of resistant genotypes collected from various breeding programmes. Except for cv Murray, which carries the Vm gene, all these resistant genotypes showed the markers OPAM19₂₂₀₀ and OPAL07₅₈₀.     

Flower morphology of the resurrection plant Chamaegigas intrepidus Dinter and some of its potential pollinators

The resurrection plant Chamaegigas intrepidus Dinter (Scrophulariaceae) is a rare endemic species growing in ephemeral rock pools on isolated granite outcrops in Central Namibia. Previous studies suggested a high degree of gene flow within individual pools. Therefore, floral morphology, pollination and potential pollinators of the plant species were studied while the plants were at full flower set.The zygomorphous, intensively scenting flowers carry dense layers of trichomes (400–1600 mm⁻²) on the lower lip, similar to well-known oil-flowers. Four species of potential pollinators could be found. Two of them the Hymenoptera, Apis mellifera and Liotrigona bottegoi, were found to be rare, whereas beetles of the genus Condylops spec. (Condylops erongoensis and a new species) showed up with numbers up to 50 individuals m⁻² in some pools, visiting the flowers most frequently. Individuals of Liotrigona and Condylops were proven to carry pollen of Chamaegigas after their flower visits. The results are discussed in relation to the genetic variability of the plant and the phenomenon of pollen limitation in rare plant species.     

Transgenic wheat progeny resistant to powdery mildew generated by Agrobacterium inoculum to the basal portion of wheat seedling

To improve the transformation efficiency of wheat (Triticum aestivum L.) mediated by Agrobacterium tumefaciens, we explored the possibility of employing the basal portion of wheat seedling (shoot apical meristem) as the explants. Three genotypes of wheat were transformed by A. tumefaciens carrying β-1, 3-glucanase gene. After vernalization, the seeds to be transformed were germinated. When these seedlings grew up to 2∼5 cm, their coleoptile and half of the cotyledon were cut out, and the basal portions were infected by A. tumefaciens. A total 27 T₀ transgenic plants were obtained, and the average transformation efficiency was as high as 9.82%. Evident segregation occurred in some of the T₁ plants, as was indicated by PCR and Southern blotting analysis. Investigation of the T₂ plants revealed that some transformed plants had higher resistance to powdery mildew than the controls. Northern blotting revealed that β-1, 3-glucanase gene was normally expressed in the T₂ plants, which showed an increased resistance to powdery mildew. The results above indicate that the exogenous gene has been successfully integrated into the genome of wheat, transmitted and expressed in the transgenic progeny. From all the results above, it can be concluded that Agrobacterium inoculum to the basal portion of wheat seedling is a highly efficient and dependable transformation method. It can be developed into a practicable method for transfer of target gene into wheat.Tong-Jin Zhao and Shuang-Yi Zhao contributed equally to this paper.     

Refugia and habitat partitioning among midges (Diptera: Chironomidae) in rain-pools

ABSTRACT.

• 1 Rain-pools on granite exposures are common in tropical Africa and they are inhabited by the larvae of two midge species, Chironomus imicola Kieffer and Chironomus pulcher Wiedemann.
• 2 These pools differ from other ephemeral island habitats such as carrion, fallen fruit, sap flows, mushrooms and dung in their high degree of spatial predictability. Most pools are predictably inhabited by either C.imicola or C.pulcher. This finding is considered in the light of contemporary competition theory.
• 3 An arrangement whereby the same species repeatedly invades the same temporary habitat indicates that there is something about the habitat itself causing the discrimination. I suggest that degree of exposure to sunlight may provide a cue allowing females to discriminate between pools as oviposition sites.
• 4 Both species survive the long dry season by retreating to pools of river water left by receding rivers. Dispersal to rain-pools by emerging adults occurs during the following season with imicola occupying rain-pools remote from shaded rivers. Appropriately, measured against pulcher, the biology of imicola is that of a colonizing species.
• 5 The possibility is discussed that the chironomids in rock-pools do not survive to breed. Instead, these populations may be maintained externally, for example by invasions from breeding populations in the Great Lakes of Africa, where larval densities may be too low to be detected by sampling. The evolutionary implications of such a situation are considered.

     

Association of gene-linked SSR markers to seed glucosinolate content in oilseed rape (Brassica napus ssp. napus)

Breeding of oilseed rape (Brassica napus ssp. napus) has evoked a strong bottleneck selection towards double-low (00) seed quality with zero erucic acid and low seed glucosinolate content. The resulting reduction of genetic variability in elite 00-quality oilseed rape is particularly relevant with regard to the development of genetically diverse heterotic pools for hybrid breeding. In contrast, B. napus genotypes containing high levels of erucic acid and seed glucosinolates (++ quality) represent a comparatively genetically divergent source of germplasm. Seed glucosinolate content is a complex quantitative trait, however, meaning that the introgression of novel germplasm from this gene pool requires recurrent backcrossing to avoid linkage drag for high glucosinolate content. Molecular markers for key low-glucosinolate alleles could potentially improve the selection process. The aim of this study was to identify potentially gene-linked markers for important seed glucosinolate loci via structure-based allele-trait association studies in genetically diverse B. napus genotypes. The analyses included a set of new simple-sequence repeat (SSR) markers whose orthologs in Arabidopsis thaliana are physically closely linked to promising candidate genes for glucosinolate biosynthesis. We found evidence that four genes involved in the biosynthesis of indole, aliphatic and aromatic glucosinolates might be associated with known quantitative trait loci for total seed glucosinolate content in B. napus. Markers linked to homoeologous loci of these genes in the paleopolyploid B. napus genome were found to be associated with a significant effect on the seed glucosinolate content. This example shows the potential of Arabidopsis-Brassica comparative genome analysis for synteny-based identification of gene-linked SSR markers that can potentially be used in marker-assisted selection for an important trait in oilseed rape. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Molecular subtyping of clinical isolates of <Emphasis Type="Italic">Candida albicans</Emphasis> and identification of <Emphasis Type="Italic">Candida dubliniensis</Emphasis>in Malaysia

The genotypes of 221 recent isolates of Candida albicans from various clinical specimens of 213 patients admitted to the University Malaya Medical Centre, Malaysia was determined based on the amplification of a transposable intron region in the 25 S rRNA gene. The analyses of 178 C. albicansisolated from nonsterile clinical specimens showed that they could be classified into three genotypes: genotype A (138 isolates), genotype B (38 isolates) and genotype C (2 isolates). The genotyping of 43 clinical isolates from sterile specimens showed that they belonged to genotype A (29 isolates), genotype B (10 isolates), genotype C (2 isolates) and genotype D (2 isolates). The overall distribution of C. albicans genotypes in sterile and nonsterile specimens appeared similar, with genotype A being the most predominant type. This study reported the identification of C. dubliniensis (genotype D) in 2 HIV-negative patients with systemic candidiasis, which were missed by the routine mycological procedure. The study demonstrated the genetic diversity of clinical isolates of C. albicans in Malaysia.     

Imputing missing yield trial data

Summary Intraspecific mitochondrial DNA (mtDNA) diversity was determined in 23 Phaseolus vulgaris genotypes, and compared to previously observed variability of morphoagronomic characters and isozyme loci. Twenty of the lines were collected from Malawian landraces; the other three were pure-bred cultivars. The mtDNAs were digested with eight restriction endonucleases, revealing complex banding patterns. Southern hybridization using cosmid clones covering about 200-kb of the genome showed a considerable amount of uniformity of the mtDNA banding patterns. However, five restriction fragment length polymorphisms (RFLPs) were detected, dividing the bean lines into two groups corresponding to the previously known Mesoamerican and Andean gene pools of P. vulgaris. The cultivar Mecosta was separated from the rest of the lines by an additional RFLP. At least two out of the six RFLPs are believed to be due to base-pair mutation events. Our results provide the first evidence that the cytoplasms of the two major germ plasm pools of beans are distinct.     

Parasites promote host gene flow in a metapopulation

Local adaptation is a powerful mechanism to maintain genetic diversity in subdivided populations. It counteracts the homogenizing effect of gene flow because immigrants have an inferior fitness in the new habitat. This picture may be reversed in host populations where parasites influence the success of immigrating hosts. Here we report two experiments testing whether parasite abundance and genetic background influences the success of host migration among pools in a Daphnia magna metapopulation. In 22 natural populations of D. magna, immigrant hosts were found to be on average more successful when the resident populations experienced high prevalences of a local microsporidian parasite. We then determined whether this success is due to parasitism per se, or the genetic background of the parasites. In a common garden competition experiment, we found that parasites reduced the fitness of their local hosts relatively more than the fitness of allopatric host genotypes. Our experiments are consistent with theoretical predictions based on coevolutionary host-parasite models in metapopulations. A direct consequence of the observed mechanism is an elevated effective migration rate for the host in the metapopulation.     

Stock Transfers in Spanish Brown Trout Populations: A Long-term Assessment

Synopsis Stocking of fish from other populations has been commonly employed for enhancement of wild brown trout, Salmo trutta, populations in north Spain. Young hatchery reared brown trout of central European origin were introduced into some Asturian rivers every year since 1984. Based on variation at the isozyme locus LDH-C1* and at the microsatellite locus BFRO 002, two genetic markers race-specific in Salmo trutta, we detected introgression of foreign genomes into native gene pools in some Spanish trout populations where only pure native individuals were present 10 years ago. We strongly suggest development of alternative management policies for conservation of Spanish natural brown trout populations without endangering the traditional recreational fisheries. Jorge I. Izquierdo, Ana G. F. Castillo: These two authors contributed equally to the article.