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.     

Cloning and expression of the α–L-arabinofuranosidase gene (ABF2) of Aspergillus niger in Saccharomyces cerevisiae

 First-strand cDNA was prepared from mRNA of Aspergillus niger MRC11624 induced on oat spelts xylan. Using the cDNA as a template, the α-L-arabinofuranosidase gene (abf B) was amplified with the polymerase chain reaction technique. The abf B DNA fragment was inserted between the yeast phosphoglycerate kinase I gene promoter (PGK1 _P ) and terminator (PGK1 _T ) sequences on a multicopy episomal plasmid. The resulting construct PGK1 _P -abf B-PGK1 _T was designated ABF2. The ABF2 gene was expressed successfully in Saccharomyces cerevisiae and functional α-L-arabinofuranosidase was secreted from the yeast cells. The ABF2 nucleotide sequence was determined and verified to encode a 449-amino-acid protein (Abf 2) that is 94% identical to the α-L-arabinofuranosidase B of A. niger N400. Maximum α-L-arabinofuranosidase activities of 0.020 U/ml and 1.40 U/ml were obtained with autoselective recombinant S. cerevisiae strains when grown for 48 h in synthetic and complex medium respectively. Received: 29 January 1996/Received revision: 3 May 1996/Accepted: 9 May 1996     

Diversity characterization and association analysis of agronomic traits in a Chinese peanut （Arachis hypogaea L.） mini-core collection

Association mapping is a powerful approach for exploring the molecular basis of phenotypic variations in plants. A peanut （Arachis hypogaea L.） mini-core collection in China comprising 298 accessions was genotyped using lo9 simple sequence repeat （SSR） markers, which identified 554 SSR alleles and phenotyped for 15 agronomic traits in three different environments, exhibiting abundant genetic and phenotypic diversity within the panel. A model-based structure analysis assigned all accessions to three groups. Most of the accessions had the relative kinship of less than o.05, indicating that there were no or weak relationships between accessions of the mini- core collection. For 15 agronomic traits in the peanut panel, generally the Q ＋ K model exhibited the best performance to eliminate the false associated positives compared to the Q model and the general linear model-simple model. In total, 89 SSR alleles were identified to be associated with 15 agronomic traits of three environments by the Q＋K model-based association analysis. Of these, eight alleles were repeatedly detected in two or three environments, and 15 alleles were commonly detected to be associated with multiple agronomic traits. Simple sequence repeat allelic effects confirmed significant differences between different genotypes of these repeatedly detected markers. Our results demonstrate the great potential of integrating the association analysis and marker-assisted breeding by utilizing the peanut mini-core collection.     

Identification and Molecular Characterization of Homeologous Δ9-Stearoyl Acyl Carrier Protein Desaturase 3 Genes from the Allotetraploid Peanut (Arachis hypogaea)

The stearoyl–acyl carrier protein (ACP) desaturase (SAD) is a nuclear-encoded, plastid-localized soluble desaturase that catalyzes the conversion of stearoyl-ACP to oleoyl-ACP and plays a key role in the determination of the properties of the majority of cellular glycerolipids. Sad genes from a variety of plant species have been cloned and characterized. However, in peanut (Arachis hypogaea), an important edible and oilseed crop, these genes have not yet been characterized. By searching peanut expressed sequence tag (EST) and parallel sequencing (454) libraries, we have identified three members of the ahSad gene family. Among them, only one gene, ahSad3, was exclusively expressed during seed development and in a manner fully corresponding to oil accumulation. Both ahSad3 homeologous genes (ahSad3A and ahSad3B) were recovered from the allotetraploid peanut, and their mRNA expression levels were characterized. The open reading frames for ahSad3A and ahSad3B are 98% identical and consist of 1,158 bp, encoding a 386-full-amino-acid protein, with one intron in the coding sequence. Comparisons of the sequences of these two homeologous genes revealed seven single-nucleotide polymorphisms and one triplet insertion in the coding region. Southern blot analysis indicated that there are only two copies of the ahSad3 gene in the peanut genome. Homeolog-specific gene expression analysis showed that both ahSad3 homeologs are expressed in developing seeds, but gene expression is significantly biased toward the B genome. Our results point to ahSad3 as a possible target gene for manipulation of fatty acid saturation in A. hypogaea.     

Isolation and characterization of the Δ12-fatty acid desaturase in peanut (Arachis hypogaea L.) and search for polymorphisms for the high oleate trait in Spanish market-type lines

An understanding of the molecular mechanisms that are responsible for increased oleic acid accumulation would open avenues to alter peanut fatty acid composition and allow detection of polymorphic regions which can be used for marker assisted selection (MAS). Δ¹²-Fatty acid desaturase (FAD) was isolated and characterized from genotypes having a low or high oleic to linoleic acid O/L ratio – genotypes, Tamspan 90 (T-90) and F435–2-2 (F435), respectively. Southern blots showed three to four copies per haploid genome, and no major differences in organization between the two parental lines. Approximately 3525 bp was isolated from both genotypes, including a genomic walk toward the promoter region. The Δ¹²-Fad contains a putative intron, the coding region at the 3′ end, and an open reading frame (ORF) of 1140 bp encoding 379 amino acids. Comparisons of the coding sequences from the high and low oleic acid genotypes revealed several single nucleotide polymorphisms (SNPs). Two polymorphisms appear to be associated with the high O/L trait. The first is an ”A” insertion 442 bp after the start codon. The ”A” insertion shifts the amino acid reading frame, probably resulting in a truncated, inactive protein and the loss of one of three histidine boxes believed to be involved in metal ion complexation required for the reduction of oxygen. Another polymorphism at 448 bp from the start codon results in an amino acid change. The region containing the polymorphisms was amplified from leaf tissue of several independently derived backcross lines (IDBLs). Most high O/L lines had either the ”A” insertion or the amino acid substitution. Received: 1 February 2000 / Accepted: 20 March 2000     

Production of eicosapentaenoic acid (EPA, 20:5n-3) in transgenic peanut (Arachis hypogaea L.) through the alternative Δ8-desaturase pathway

Molecular Biology Reports - An important alternative source of fish oil is its production by plants through metabolic engineering. To produce eicosapentaenoic acid (EPA, 20:5n-3) in peanut through...     

The partial acid hydrolysis of a highly dextrorotatory fragment of the cell wall of Aspergillus niger. Isolation of the α-(1→3)-linked dextrin series

1. The cell-wall composition of Aspergillus niger has been investigated. Analysis shows the presence of six sugars, glucose, galactose, mannose, arabinose, glucosamine and galactosamine, all in the d-configuration, except that a small amount of l-galactose may be present. Sixteen common amino acids are also present. 2. The wall consists chiefly of neutral carbohydrate (73-83%) and hexosamine (9-13%), with smaller amounts of lipid (2-7%), protein (0.5-2.5%) and phosphorus (less than 0.1%). The acetyl content (3.0-3.4%) corresponds to 1.0mole/mole of hexosamine nitrogen. 3. A fractionation of the cell-wall complex was achieved, with or without a preliminary phenol extraction, by using n-sodium hydroxide. Though this caused some degradation, 30-60% of the wall could be solubilized (depending on the preparation). Analyses on several fractions suggest that fractionation procedures bring about some separation of components although not in a clear-cut fashion. 4. Cell-wall preparations were shown to yield a fraction having [alpha](D) approx. +240 degrees (in n-sodium hydroxide) and consisting largely of glucose. This was separated into two subfractions, one of which had [alpha](D)+281 degrees (in n-sodium hydroxide) and had properties resembling the polysaccharide nigeran; the other had [alpha](D) +231 degrees (in n-sodium hydroxide). It is suggested that nigeran is a cell-wall component.     

β-Glucan hydrolases from Aspergillus niger. Isolation of a β-(1→4)-glucan hydrolase and some properties of the β-(1→3)-glucan-hydrolase components

1. The components of an enzyme preparation from Aspergillus niger, which hydrolysed substrates containing beta-(1-->3)- and beta-(1-->4)-glucosidic linkages, were separated by calcium phosphate and Dowex 1 column chromatography. 2. The hydrolytic activity of each fraction from both types of column towards laminaribiose, laminarin, carboxymethylpachyman, pachydextrins, salicin, cellobiose, cellopentaose and swollen cellulose was tested. 3. The activity towards the beta-(1-->3)-glucosidic substrates was found in three well-separated groups of fractions. The differences in action pattern of these groups is discussed. 4. Preparative-scale chromatography that enabled the separation of a beta-(1-->4)-glucan-glucanohydrolase component substantially free of activity towards beta-(1-->3)-glucosidic substrates is described. Residual beta-(1-->3)-glucan-hydrolase activity was removed by adsorption on to insoluble laminarin at pH3.5.     

Cellulolytic complex of Aspergillus niger under conditions for citric acid production. Isolation and characterization of two β-(1→4)-glucan hydrolases

Summary Two -(14)-endoglucanases have been purified from industrial waste broth of Aspergillus niger grown under conditions which produce citric acid. Molecular weights for endoglucanase A were 43,000 and 25,000 for endoglucanase B. Both enzymes exhibited very similar properties: a rather broad pH optimum between pH 2 and 7 for CM-cellulose hydrolysis and an inability to degrade crystalline cellulose. The endoglucanases have a higher thermal stability at acid pH (up to 60°C) than at alkaline pH. They are inhibited by iodine, HgCl₂ and N-bromosuccinimide.     

Differences in the floral anthocyanin content of violet–blue flowers of Vinca minor L. and V. major L. (Apocynaceae)

Two novel delphinidin 3-(tri or di)-glycoside-7-glycosides were isolated from the violet–blue flowers of Vinca minor L. and V. major L. (Family: Apocynaceae), and determined to be delphinidin 3-O-[2-O-(β-xylopyranosyl)-6-O-(α-rhamnopyranosyl)-β-galactopyranoside]-7-O-(α-rhamnopyranoside) [= delpphinidin 3-(2^G-xylosylrobinobioside)-7-rhamnoside] as major floral anthocyanin of V. minor and delphinidin 3-O-[6-O-(α-rhamnopyranosyl)-β-galactopyranoside]-7-O-(α-rhamnopyranoside) [= delpphinidin 3-robinobioside-7-rhamnoside] as major floral anthocyanin of V. major by chemical and spectroscopic methods. In addition, chlorogenic acid and kaempferol 3-O-[6-O-(α-rhamnopyranosyl)-β-galactopyranoside]-7-O-(α-rhamnopyranoside) [= kaempferol 3-robinobioside-7-rhamnoside (robinin)] were identified in these flowers. In this paper, the relation between the structure of floral anthocyanins and classification of Vinca species was discussed.     

Proposal of Spinulacorpus biforme (Smales, 2014) n. g., n. comb. and the Spinulacorpidae n. fam. to resolve paraphyly of the acanthocephalan family Rhadinorhynchidae Lühe, 1912

Systematic Parasitology - Previous phylogenetic analyses of the Acanthocephala have demonstrated that the families Rhadinorhynchidae Lühe, 1912 and Transvenidae Pichelin & Cribb, 2001...     

Identification of the initial binding sites of αs2-casein f(183-207) and effect on bacterial membranes and cell morphology

The aim of this work was to identify the initial binding sites to the bacterial membranes of the antimicrobial peptide α_s2-casein f(183-207) and also to acquire further insight into membrane permeabilization of this peptide. Furthermore, cell morphology was studied by transmission electron microscopy. In all the experiments, bovine LFcin was employed as a comparison. Results showed that initial binding sites of α_s2-casein f(183-207) peptide were lipoteichoic acid in Gram-positive bacteria and lipopolysaccharide in Gram-negative. The peptide was able to permeabilize the outer and inner membranes. Moreover, the α_s2-casein peptide f(183-207) generated pores in the outer membrane of Gram-negative bacteria and in the cell wall of Gram-positive bacteria. In the Gram-negative bacteria, f(183-207) originated cytoplasm condensation, and in the Gram-positive bacteria the cytoplasmic content leaked into the extracellular medium. Furthermore, the experiments of inner and outer membrane permeabilization performed with LFcin-B showed that this peptide also has the ability to permeabilize both the inner and outer membranes.     

In vitro expression of genes affecting whole plant phenotype — the effect of Rht/Gai alleles on the callus culture response of wheat (Triticum aestivum L. em. Thell)

Summary Calli were initiated from immature embryos of 12 lines of hexaploid wheat (Triticum aestivum L. em. Thell). The lines were from 3 varieties — April Bearded, Bersee and Maris Huntsman — isogenic for the reduced height/gibberellic acid insensitivity (Rht) genes — Rht1, Rht2 and Rht3 — and the tall (rht) allele. The dwarfing genes had significant effects on the growth and morphogenesis of calli. The genes interacted with the 2,4-D in the medium and the varietal background. Calli of each line were cultured in the presence and absence of 1 mg/l of gibberellic acid (GA), but there was no interaction of the Rht genes with GA in vitro. The effect of the Rht genes is discussed in relation to their effects on cellular hormone metabolism and their involvement in previously described chromosome 4B effects in culture.     

4″-O-(ω-Quinolylamino-alkylamino)propionyl derivatives of selected macrolides with the activity against the key erythromycin resistant respiratory pathogens

Four macrolides—6-O-methyl-8a-aza-8a-homoerythromycin, clarithromycin, azithromycin and azithromycin 11,12-cyclic carbonate, have been selected for the construction of a series of new quinolone derivatives. The quinolone moiety is connected to the macrolide scaffold via a diaminoaklyl 4″-O-propionyl ester chain of varying length. At the terminus the linker is attached via one of the nitrogen atoms in the linker at C(6) or C(7) of the quinolone. Many of compounds described, particularly clarithromycin derivative 37, and azithromycin derivatives 48 and 55, exhibited excellent antibacterial activity against a wide range of clinically relevant macrolide-resistant organisms, with profiles superior to that of telithromycin, an enhanced spectrum ketolide.     

Comparative study of the antitumor effect of two types of murine recombinant interferons, (β) and (γ), against B16-F10 melanoma

Summary A comparative study of the antitumor effect of murine recombinant interferon() Mu-rIFN() and murine recombinant interferon() Mu-rIFN() on B16-F10 melanoma was conducted. Administration of Mu-rIFN() i.p. into C57BL/6 mice on days 1 to 7 produced a higher suppressive effect than Mu-rIFN() both on the growth of s.c. implanted tumor and on the formation of artificial pulmonary metastasis. Pharmacokinetic study of Mu-rIFN() demonstrated that high plasma levels were retained for a long time. In clonogenic assay, Mu-rIFN() at 1000 units/ml showed about 80% inhibition of colonies of B16-F10 melanoma. However, Mu-rIFN() hardly inhibited the colonies, even at 1000 units/ml. Augmentation of natural killer (NK) cytotoxicity was much greater with Mu-rIFN() than Mu-rIFN(), whereas Mu-rIFN() enhanced the cytotoxicity of peritoneal macrophages more strongly than Mu-rIFN(). Injection of Mu-rIFN() i.p. 1 day before tumor challenge also inhibited the formation of pulmonary metastasis of B16-F10 melanoma. However, pretreatment of mice with carrageenan significantly suppressed the inhibitory effect of Mu-rIFN(). From these results, it is suggested that the inhibitory effect of Mu-rIFN() on the tumor growth and metastases of B16-F10 melanoma is mediated partly by direct antitumor effect and partly by the activation of macrophages, and that the augmentation of NK activity contributes mainly to the antitumor effect of Mu-rIFN().     

Water stress and the catabolism of (±)-abscisic acid by excised leaves of Hordeum vulgare L. cv. Dyan

When excised, light-grown leaves of Hordeum vulgare were fed with (±)-[2-¹⁴C]-abscisic acid and stressed until they had lost 12% of their original fresh weight, marked changes in the distribution of radioactivity between abscisic acid and its catabolites were observed. Wilted leaves were less able than their turgid counterparts to transform (±)-[2-¹⁴C]-abscisic acid into 2-hydroxymethyl abscisic acid, dihydrophaseic acid and water-soluble conjugates of abscisic acid. Water stress had little effect on the production of phaseic acid although refeeding studies with [¹⁴C]-phaseic acid showed that the step from phaseic acid to dihydrophaseic acid was inhibited in wilted leaves. Evidence was obtained which suggested that these changes did not result from dilution of applied, radiolabelled substrate by endogenous abscisic acid. The catabolites of (±)-abscisic acid were identified by capillary gas chromatography-mass spectrometry.     

Abundance and aggregation egg of Aedes aegypti L. and Aedes albopictus (Skuse) (Diptera: Culicidae) in the north and northwest of the State of Paraná, Brazil

The abundance and aggregation of eggs of Aedes aegypti L. and Aedes albopictus Skuse was evaluated in the municipalities of Cambé, Ibipor?, Jacarezinho, Maringá and Paranavaí, in the State of Paraná, Brazil by means of oviposition traps. Of the 225 installed traps, 100 were registered as positive for eggs; 4140 eggs were collected, thus demonstrating an highly aggregate distribution. Both species were registered in Cambé, Jacarezinho, Maringá and Paranavaí. Ae. albopictus was generally less abundant and was not present in Ibipor? nor in the oviposition traps of a second collection of Maringá. The relation between sexes for Ae. aegypti was approximately 1:1. In the comparison of the number of adults collected between the two species, a negative correlation was obtained in the samples of Maringá and Cambé, what was attributed the seasonality of these populations. The coexistence of these species indicates that both are under pressure by the control programs, therefore specific evaluations are necessary.     

The responses of Ligia oceanica (L.) (Crustacea: Isopoda) to infection by Maritrema linguilla Jäg. 1908 and Microphallus similis (Jäg. 1900) (Digenea: Microphallidae) and to abiotic implants

The initial and principal encapsulation response of Ligia oceanica to Araldite implants and to encysted metacercariae of Maritrema linguilla is hemolymph coagulation followed by limited hemocyte agglutination. Granules secreted by isolated granulocytes and semigranulocytes may catalyze coagulation. Isolated hyaline cells explode and make an insignificant contribution to the initial cyst wall. Later, hemocytes agglutinate and some granulocytes retain their granules which become melanized. Eventually, a wide multilayered hos capsule is formed. Unencysted metacercariae of M. linguilla transplanted from the pleopods into the dorsal hemocoel of another specimen of L. oceanica encyst and become encapsulated but are not damaged by encapsulation. Transplanted encysted metacercariae are also encapsulated and unharmed. Cercariae implanted directly into the dorsal hemocoel, however, fail to encyst, become encapsulated, die, and lyse within the capsule. Implanted cercariae and encysted metacercariae of Microphallus similis are also encapsulated and destroyed in the hemocoel of L. oceanica. The absence of host response to the naturally infecting unencysted parasite in the pleopod sinuses may be attributed to rhythmic movement, mucopolysaccharide secretions and to the retention of excreta within the excretory bladder. Once the excreta is released during cyst formation in the dorsal hemocoel, encapsulation occurs but this does not appear to harm the parasite. On the contrary, considerable growth occurs within the cyst which suggests that the parasite may absorb nutrients released from necrotic hemocytes.