A comparative analysis of biparental mating and selfing in pearl millet (Pennisetum typhoïdes) S & H

Summary A study was made of the nature of variation and changes in association for characters related to yield, development and disease resistance in two sets of progenies one set derived through biparental mating (A-series) and the other by traditional inbreeding (selfing) from the F₂ onwards (T-series), in a 22 × 22 diallel of dwarf derivatives of Pennisetum typhoides S & H. Comparison of means and variances within and between the two groups of progenies (A and T-series) and with those of released hybrids (HB-1, HB-2, HB-3, HB-4) for seventeen characters (six related to development, six to productivity, three to vegetative growth and one each to earliness and disease incidence) showed significant variation among the lines of the two series for synchrony of tillering, earliness, disease incidence, chlorophyll depth, grain hairiness, tiller number, plant height, length of earhead, grain yield and grain weight. The A-series selections were significantly superior to the T-series for most of the characters, indicating the effectiveness of selection in biparental matings compared with selection in selfed progenies for these traits. Many of the A-series (220 out of 800) selections were also superior to the existing released hybrids.Changes in the magnitude and nature of association among twelve important characters in the two populations were more favourable in the biparental matings: of 66 possible correlation coefficients examined, 25 cases were found with changes in favour of selections from biparental matings (A-series). These changes were probably due to the breakage of unfavourable linkages and changes in phase of linkage, and indicate the usefulness of biparental matings in the simultaneous improvement of the population for several attributes.     

Creatine kinase from the electric organ of Electrophorus electricus (L.)—isozyme analysis

• 1.1. Creatine kinase (CPK) isozymes of extracts from the electric organ, dorsal muscle and brain of Electrophorus electricus (L.) were analysed with Cellogel electrophoresis. A single component corresponding to the MB-form was obtained for both electric organ and the dorsal muscle. The BB-form was present in the brain extract.
• 2.2. Upon acetone fractionation of the aqueous extract of electric organ, the final fraction was submitted to gel filtration and presented a single peak of CPK activity.
• 3.3. Characterization of this fraction by thin-layer gel filtration indicated an apparent molecular weight of 80,000 which corresponds to the enzyme dimeric structure.
• 4.4. The implications of this finding with the muscular origin of the electric organ are discussed.

     

Genetic structure of sable (Martes zibellina L.) in Eurasia—analysis of the mitochondrial lineages distribution

The phylogeography of the sable, which is a commercially valuable species, is extremely complicated and poorly investigated. Specifically, the effects of factors such as the range dynamics of the sable during the Pleistocene Epoch, the localization of glacial refugia, species distribution pattern in Holocene, and recent dramatic population decline, along with massive reacclimatization measures, on the species phylogeography remain unclear. Based on the sequence analysis of the control region of mitochondrial DNA from sables that inhabit different parts of the species range, a suggestion was made of the considerably high Pleistocene genetic diversity in sable, which was subsequently lost. The initial diversity of mitochondrial lineages is mostly preserved in the Urals, while in the eastern part of the range, it seems to have been depleted as early as before the last glacial maximum. On the other hand, the even greater depletion of the mitochondrial lineages observed in some populations of central Siberia can be associated with the dramatic population decline at the turn of the 20th century.     

Isolation and separation of α-amylase inhibitors I-1 and I-2 from seeds of ragi (Indian finger millet, Eleusine coracana) by metal chelate affinity precipitation

The concept of immobilized metal affinity chromatography (IMAC) was integrated with affinity precipitation for the single step isolation of -amylase inhibitors I-1 and 1-2 from the seeds of ragi (Indian finger millet, Eleusine coracana). -Amylase inhibitor I-1 was purified 13-fold with a yield of 84%, using Cu(II) loaded thermosensitive metal chelate copolymer of N-isopropylacrylamide (NIPAM) and 1-vinyl imidazole (VI). The protein also showed trypsin inhibitory activity. The binding of the protein to the copolymer was strongly pH dependent. -Amylase inhibitor I-2 was recovered in the supernatant as unprecipitated protein with significant purification and constituted 27% of the total inhibitor power. The yield with respect to inhibitor I-2 was around 85%. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed significant purification of inhibitor I-1 and indicated evident separation of the two proteins on metal chelate affinity precipitation.     

Using defined finger–finger interfaces as units of assembly for constructing zinc-finger nucleases

Zinc-finger nucleases (ZFNs) have been used for genome engineering in a wide variety of organisms; however, it remains challenging to design effective ZFNs for many genomic sequences using publicly available zinc-finger modules. This limitation is in part because of potential finger–finger incompatibility generated on assembly of modules into zinc-finger arrays (ZFAs). Herein, we describe the validation of a new set of two-finger modules that can be used for building ZFAs via conventional assembly methods or a new strategy—finger stitching—that increases the diversity of genomic sequences targetable by ZFNs. Instead of assembling ZFAs based on units of the zinc-finger structural domain, our finger stitching method uses units that span the finger–finger interface to ensure compatibility of neighbouring recognition helices. We tested this approach by generating and characterizing eight ZFAs, and we found their DNA-binding specificities reflected the specificities of the component modules used in their construction. Four pairs of ZFNs incorporating these ZFAs generated targeted lesions in vivo, demonstrating that stitching yields ZFAs with robust recognition properties.     

Coleoptile length,gibberellin sensitivity and concentrations in five non-allelic dwarf mutants of pearl millet — Pennisetum glaucum (L.) R. Br.

The concentrations of endogenous gibberellins (GAs) were determined by combined gas chromatography-mass spectrometry in shoots of five non-allelic dwarfs of pearl millet Pennisetum glaucum (L.) R. Br. One mutant (d3), with an extreme dwarf phenotype, was found to be deficient in all GAs measured; the others (d1, d2, d4 and the quantitatively inherited dwarf) had similar levels of GAs to the tall genotype. Only the GA-deficient dwarf recovered the tall phenotype in response to applying GA₃ up to the adult stage, while the others showed slight to moderate responses at the seedling stage, depending on the season, and no response at later stages. The d1, d3 and d4 dwarfs had short coleoptiles. A wide range of coleoptile lengths with a normal distribution pattern was observed in the tall, d2 and the quantitatively inherited dwarf, suggesting that there is polygenic control of this trait.     

Differentiation with drift: a spatio-temporal genetic analysis of Galápagos mockingbird populations (Mimus spp.)

Small and isolated island populations provide ideal systems to study the effects of limited population size, genetic drift and gene flow on genetic diversity. We assessed genetic diversity within and differentiation among 19 mockingbird populations on 15 Galápagos islands, covering all four endemic species, using 16 microsatellite loci. We tested for signs of drift and gene flow, and used historic specimens to assess genetic change over the last century and to estimate effective population sizes. Within-population genetic diversity and effective population sizes varied substantially among island populations and correlated strongly with island size, suggesting that island size serves as a good predictor for effective population size. Genetic differentiation among populations was pronounced and increased with geographical distance. A century of genetic drift did not change genetic diversity on an archipelago-wide scale, but genetic drift led to loss of genetic diversity in small populations, especially in one of the two remaining populations of the endangered Floreana mockingbird. Unlike in other Galápagos bird species such as the Darwin''s finches, gene flow among mockingbird populations was low. The clear pattern of genetically distinct populations reflects the effects of genetic drift and suggests that Galápagos mockingbirds are evolving in relative isolation.     

Risk analysis for biological weed control – simulating dispersal of Sclerotinia sclerotiorum (Lib.) de Bary ascospores from a pasture after biological control of Cirsium arvense (L.) Scop.

Biological control of Cirsium arvense(L.) Scop. in pasture by the plurivorous plantpathogenic fungus Sclerotiniasclerotiorum (Lib.) de Bary mayresult in the formation, escape and aerialdispersal of ascospores, creating an additionaldisease risk in down-wind market garden crops. To determine the width of a safety zone for apasture subjected to this form of weed control,we simulated the spatial pattern in the ratioof added (due to biocontrol) to naturallyoccurring airborne ascospores (due to marketgarden crops) around a 1ha virtual biocontrolpasture under either sheep or dairy cattlegazing over a 91-day emission period in 1996 inCanterbury, New Zealand. This was achievedusing a unique combination of two computermodels; SPORESIM-1D (for spore escape from avegetation source) and PC-STACKS (a modernGaussian plume model for dispersal beyond asource). Plumes of dispersing ascospores weremodelled for each hour of the emission periodfor both the virtual market garden andbiocontrol sites, and the aerial density of theascospores was averaged over the period. Assuming that a 1:1 ratio of added to naturallypresent spores is acceptable, no safety zonewas necessary for either of the modeledpastures. A ten-fold ratio (1:10 added tonatural) necessitated safety zones of 300 and150 m for the sheep and dairy pasturerespectively. Uncertainties associated withextrapolation of this conclusion to individualpasture management scenarios, and to otheryears and climatically different regions arediscussed.     

Phylogenetic relationship of wild and cultivated yam species (Dioscorea spp.) of India inferred from PCR–RFLP analysis of two cpDNA loci

The genus Dioscorea comprises approximately 600 species worldwide and about 50 in India. Dioscorea is economically very useful because of its food and medicinal value. Polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) analysis was employed to study interspecific relationship between four cultivated and thirteen wild species belonging to seven taxonomical sections found in India. Restriction digestion analysis was performed for the amplified products of chloroplast DNA (cpDNA) loci psbA–trnH and trnL-F regions. Out of 17 restriction endonucleases tested, 5 endonucleases had restriction sites in psbA–trnH region and 6 endonucleases had restriction sites in trnL-F region. Digestion of amplified products with these restriction endonucleases showed 70 fragments of which 62 fragments (88 %) were polymorphic. Dendrogram obtained from UPGMA cluster analysis of two combined loci shows D. bulbifera of section Opsophyton to be closely placed with species of sect. Enantiophyllum. High similarity values obtained in this study between D. pentaphylla (sect. Botrysicyos) and D. esculenta (sect. Combilium) support their common origin. The average of genetic similarities between the ten species of Enantiophyllum section was 82.3 % showing their closeness. The present study suggests that the less studied New World species, D. floribunda (sect. Heterostemon) could have evolved from old world section, Lasiophyton. The principal component analysis (PCA) obtained from data of the two loci shows D. deltoidea of sect. Stenophora to be an outgroup. Interspecific genetic similarities showed D. deltoidea with low similarity values ranging from 27.5 to 37.5 % compared with other species and was, therefore, the most divergent species. D. deltoidea of section Stenophora shows distinctness because of its primitive characters. This study, therefore, establishes species relationships between wild and cultivated yams of India which would help in yam crop improvement programmes.     

Population structure and marker�Ctrait association analysis of the US peanut (Arachis hypogaea L.) mini-core collection

Peanut (Arachis hypogaea L.) is one of the most important oilseed and nutritional crops in the world. To efficiently utilize the germplasm collection, a peanut mini-core containing 112 accessions was established in the United States. To determine the population structure and its impact on marker-trait association, this mini-core collection was assessed by genotyping 94 accessions with 81 SSR markers and two functional SNP markers from fatty acid desaturase 2 (FAD2). Seed quality traits (including oil content, fatty acid composition, flavonoids, and resveratrol) were obtained through nuclear magnetic resonance (NMR), gas chromatography (GC), and high-performance liquid chromatography (HPLC) analysis. Genetic diversity and population structure analysis identified four major subpopulations that are related to four botanical varieties. Model comparison with different levels of population structure and kinship control was conducted for each trait and association analyses with the selected models verified that the functional SNP from the FAD2A gene is significantly associated with oleic acid (C18:1), linoleic acid (C18:2), and oleic-to-linoleic (O/L) ratio across this diverse collection. Even though the allele distribution of FAD2A was structured among the four subpopulations, the effect of FAD2A gene remained significant after controlling population structure and had a likelihood-ratio-based R ( 2 ) (R ( LR ) ( 2 ) ) value of 0.05 (oleic acid), 0.09 (linoleic acid), and 0.07 (O/L ratio) because the FAD2A alleles were not completely fixed within subpopulations. Our genetic analysis demonstrated that this peanut mini-core panel is suitable for association mapping. Phenotypic characterization for seed quality traits and association testing of the functional SNP from FAD2A gene provided information for further breeding and genetic research.     

Inheritance of downy mildew resistance, β-1,3-glucanases and peroxidases in pearl millet [Pennisetum glaucum (L.) R. Br.] crosses

The inheritance of resistance to downy mildew disease and the defense-related enzymes β-1,3-glucanase and peroxidase was studied in crosses of pearl millet using a generation-mean analysis. The study material comprised six generations (susceptible and resistant parents, F₁, F₂, BC₁ and BC₂) in three crosses. Seedlings from these generations were inoculated with the downy mildew pathogen Sclerospora graminicola and disease incidence was recorded. Analysis of constitutive levels of β-1,3-glucanase and peroxidase in the seedlings of different generations indicated that the resistant populations showed higher enzyme activities, while lower activities of the enzymes were recorded in the susceptible populations. In the generation-mean analysis, the significance of scaling tests revealed the existence of non-allelic interactions in the inheritance of resistance to downy mildew as well as with the enzymes. Among the gene effects, both additive and dominant effects were significant. All the non-allelic interaction effects were significant in the crosses. Studies on the isozyme patterns of the enzymes substantiated the results of the disease-incidence experiments in most of the generations. The results indicated that the inheritance of downy mildew disease resistance and the expression of β-1,3-glucanase and peroxidase in pearl millet is not only under the control of additive and dominant genes but are also governed by complex non-allelic interactions. Received: 30 April 2000 / Accepted: 17 October 2000     

Behaviour of a ‘sticky-desynaptic’ mutant in pearl millet

In the progeny after selfing of a normally open pollinated variety (L.S. 326-3) of pearl millet (Pennisetum americanum (L.) Leeke, n=7) one plant exhibited desynapsis, chromosome stickiness and high sterility. Meiosis was normal until diplotene. Thereafter, it was characterized by dissociation of bivalents into univalents and formation of nonspecific congregations of chromosomes at diakinesis, shrinkage of cytoplasm and occurrence of unoriented sticky chromatin masses at metaphase I, relaxation of stickiness, unbalanced chromosome numbers at the poles and laggards at anaphase I, and presence of other irregularities in subsequent stages. Meiosis was completed. Male and female sterility was high. This meiotic mutant thus has multiple effects and is inherited as a monogenic recessive and designated as st.     

Emission of hydrogen sulfide by twigs of coniferes — acomparison of Norway spruce (Picea abies (L.) Karst.), Scotch pine (Pinus sylvestris L.) and Blue spruce (Picea pungens Engelm.)

The emission of reduced volatile sulfur compounds from twigs of Norway spruce (Picea abies (L.) Karst.) was measured in the field by cryosampling and gaschromatographic analysis. Trees were growing in the Erzgebirge (E-Germany) at Oberbärenburg and at the Kahleberg and at a third stand in NW-Bavaria (S-Germany). Emission rates were also measured for Scotch pine (Pinus sylvestris L.) and Blue spruce (Picea pungens Engelm.) at the Kahleberg. Twigs still attached to the trees were enclosed in a flow-through gas exchange cuvette. H₂S was detected as the predominant reduced sulfur compound emitted from the twigs. The mean H₂S emission rate from twigs of Norway spruce varied between 0.04 pmol kg^-1 dw s^-1 at Würzburg and 6.21 pmol kg^-1 dw s^-1 at the Kahleberg. Comparing different species at the Kahleberg, the mean H₂S emission rate was almost the same from twigs of Norway spruce (6.2 pmol kg^-1 dw s^-1) and Blue Spruce trees (5.9 pmol kg^-1 dw s^-1) but it was approximately 18 times higher for Scotch pine (110 pmol kg^-1 dw s^-1). The percentage of SO₂-exclusion via H₂S-emission of the tree species investigated at the Kahleberg is calculated on the basis of data on SO₂ fluxes. It is very small for Norway spruce and Blue spruce. However, for Scotch pine, H₂S emission contributes about 10% to the detoxification of SO₂.     

Diagnostic model of saliva peptide finger print analysis of primary Sj?gren's syndrome patients by using weak cation exchange magnetic beads

Saliva diagnostics has become an attractive field utilizing nanotechnology and molecular technologies for pSS (primary Sjögren''s syndrome). However, no specific methods have been established. To refine the diagnostic power of the saliva peptide finger print for the early detection of pSS, we screened the expression spectrum of salivary peptides in pSS patients by using mass spectrometry MALDI-TOF-MS (matrix-assisted laser-desorption ionization-time-of-flight MS) combined with magnetic bead. The present study was comprised 12 pSS patients and 13 healthy controls and broken down to two different phases. In the initial ‘exploratory phase’, we enrolled seven pSS patients with eight age- and sex-matched healthy volunteers. Proteomics analysis of the unstimulated salivary samples was conducted to generate proportional peptide mass fingerprints. A diagnostic model was established. The testing cohort of the second ‘validation phase’ was represented by five pSS patients and five age- and sex-matched healthy controls. The diagnostic power of this diagnostic panel was then validated. The results showed seven m/z (mass-to-charge) ratio peaks with significant differences. Five peptides were up-regulated and two down-regulated in the pSS patients compared with matched healthy subjects. In the validation phase, four out of five pSS patients were diagnosed as pSS, and four of the five healthy controls were diagnosed as healthy controls, respectively. Potential biomarkers were also primarily predicted. The novel diagnostic proteomic model with m/z peaks 1068.1 Da, 1196.2 Da, 1738.4 Da, 3375.3 Da, 3429.3 Da, 3449.7 Da and 3490.6 Da is of certain value for early diagnosis of pSS.     

Are Early Somatic Embryos of the Norway Spruce (Picea abies (L.) Karst.) Organised?

Background

Somatic embryogenesis in conifer species has great potential for the forestry industry. Hence, a number of methods have been developed for their efficient and rapid propagation through somatic embryogenesis. Although information is available regarding the previous process-mediated generation of embryogenic cells to form somatic embryos, there is a dearth of information in the literature on the detailed structure of these clusters.

Methodology/Principal Findings

The main aim of this study was to provide a more detailed structure of the embryogenic tissue clusters obtained through the in vitro propagation of the Norway spruce (Picea abies (L.) Karst.). We primarily focused on the growth of early somatic embryos (ESEs). The data on ESE growth suggested that there may be clear distinctions between their inner and outer regions. Therefore, we selected ESEs collected on the 56^th day after sub-cultivation to dissect the homogeneity of the ESE clusters. Two colourimetric assays (acetocarmine and fluorescein diacetate/propidium iodide staining) and one metabolic assay based on the use of 2,3,5-triphenyltetrazolium chloride uncovered large differences in the metabolic activity inside the cluster. Next, we performed nuclear magnetic resonance measurements. The ESE cluster seemed to be compactly aggregated during the first four weeks of cultivation; thereafter, the difference between the ¹H nuclei concentration in the inner and outer clusters was more evident. There were clear differences in the visual appearance of embryos from the outer and inner regions. Finally, a cluster was divided into six parts (three each from the inner and the outer regions of the embryo) to determine their growth and viability. The innermost embryos (centripetally towards the cluster centre) could grow after sub-cultivation but exhibited the slowest rate and required the longest time to reach the common growth rate. To confirm our hypothesis on the organisation of the ESE cluster, we investigated the effect of cluster orientation on the cultivation medium and the influence of the change of the cluster’s three-dimensional orientation on its development. Maintaining the same position when transferring ESEs into new cultivation medium seemed to be necessary because changes in the orientation significantly affected ESE growth.

Conclusions and Significance

This work illustrated the possible inner organisation of ESEs. The outer layer of ESEs is formed by individual somatic embryos with high metabolic activity (and with high demands for nutrients, oxygen and water), while an embryonal group is directed outside of the ESE cluster. Somatic embryos with depressed metabolic activity were localised in the inner regions, where these embryonic tissues probably have a very important transport function.     

Expression,purification, and characterization of N-terminal His-tagged proteins with mutations in zinc finger 3 of zinc finger protein ZNF191(243–368)

Abstract

Zinc finger protein ZNF191(243–368), the zinc finger region of ZNF191, is potentially associated with cell proliferation in hepatocellular carninoma. A His-tag expression system was used to express and purify proteins with mutations in the zinc finger 3 of ZNF191(243–368) for analysis of protein properties, structure, and functions. The purification of the His-tag fusion proteins was simpler and faster than that of the ZNF191(243–368) inclusion bodies. The properties and structures of the His-tag fusion mutant proteins were investigated using spectrographic techniques and DNA hydrolysis experiment. The His₆-tag system could be used to express ZNF191(243–368). The presence of the His₆-tag at the N-terminus of ZNF191(243–368) did not evidently affect its properties and structure. However, the site-directed mutations in zinc finger 3 affected the structure of the protein. The DNA hydrolase activity of His₆-ZF-F3/H4 suggested that four histidines in zinc finger 3 might form a structure similar to that of the active center in a hydrolase. This work reports that continuous histidines need to form a certain structure for specific functions, and provides new insights into the design of an artificial nuclease.