Genome-wide analysis of across herd <Emphasis Type="Italic">F</Emphasis> <Subscript>st</Subscript> Heterogeneity in holsteinized cattle

To form a reference population necessary for genomic selection of dairy cattle, it is important to acquire information on the genetic diversity of the base population. Our report is the first among the studies on breeding of farm animals to implement Wright’s F-statistics for this purpose. Genotyping of animals was performed using BovineSNP50 chip. In total, we genotyped 499 heifers from 13 breeding farms in the Leningrad region. We calculated Weir and Cockerham’s F_st estimate for all pairwise combinations of herds from breeding farms and the values obtained were in the range of 0.016–0.115 with the mean of 0.076 ± 0.002. Theoretical F_st values for the same pairwise combinations of herds were calculated using the ADMIXTURE program. These values were significantly (P < 0.05) higher than Weir and Cockerham’s F_st estimates and fell in the range of 0.063–0.136 with the mean of 0.100 ± 0.001. We discuss the reasons for this discrepancy between the two sets of F_st data. The obtained F_st values were used to identify reliable molecular-genetical differences between the herds. The ADMIXTURE program breaks the pool of 476 heifers into 16 subpopulations, the number of which is close to the number of herds used in the experiment. Results of the comparison between F_st values obtained using SNP markers with published data obtained on microsatellites are in support of the common opinion that microsatellite analysis results in underestimation of F_st values. On the whole, the obtained across-herd F_st values are in the range Fst data reported for cattle breeds. Results of comparison of F_st values with the data on the origin of bulls imported from different countries lead to the conclusion on the expediency of the use of F_st data to assess heterogeneity of the herds. Thus, we have demonstrated that use of F_st data provides the means to assess genetic diversity of cattle herds and is a necessary step in the formation of a reference population for dairy cattle.     

Geographic and temporal genetic structure in <Emphasis Type="Italic">Lepeophtheirus salmonis</Emphasis> from four salmon farms along the northwest and west coasts of Ireland: results from a microsatellite analysis

The marine ectoparasitic copepod of salmonids, Lepeophtheirus salmonis (Krøyer), is a major pest of farmed Atlantic salmon (Salmo salar L.) causing great economic impact. The spatial scales over which L. salmonis populations in different salmon farms are typically connected, and the temporal scales over which L. salmonis from the same farm typically undergo genetic change are largely unknown. These questions were posed in a small-scale geographic study of population structure in L. salmonis from four salmon farms, along the northwest and west coasts of Ireland, using two outgroups from Norway and Canada. The temporal stability of genetic composition was also studied in samples collected quarterly during one year from one salmon farm in Ireland. Genetic composition in L. salmonis was characterised using four nuclear microsatellites. Significant but low genetic differentiation was observed between all sites (F _ST = 0.08), with no evidence that differentiation was correlated with geographic distance. Temporal genetic differentiation was also evident (F _ST = 0.07). An analysis of all L. salmonis samples except the ones from Norway detected two separate clusters. Each cluster contained both geographical and temporal samples. These results are consistent with a population model in which L. salmonis in salmon farms along the northwest and west coasts of Ireland are not isolated, but are potentially subject to (i) localised ecological factors at the particular farm sites or (ii) selection post-settlement or a combination thereof.     

<Emphasis Type="Italic">C7</Emphasis>-prenylation of tryptophanyl and <Emphasis Type="Italic">O</Emphasis>-prenylation of tyrosyl residues in dipeptides by an <Emphasis Type="Italic">Aspergillus terreus</Emphasis> prenyltransferase

During our search for novel prenyltransferases, a putative gene ATEG_04218 from Aspergillus terreus raised our attention and was therefore amplified from strain DSM 1958 and expressed in Escherichia coli. Biochemical investigations with the purified recombinant protein and different aromatic substrates in the presence of dimethylallyl diphosphate revealed the acceptance of all the tested tryptophan-containing cyclic dipeptides. Structure elucidation of the main enzyme products by NMR and MS analyses confirmed the attachment of the prenyl moiety to C-7 of the indole ring, proving the identification of a cyclic dipeptide C7-prenyltransferase (CdpC7PT). For some substrates, reversely C3- or N1-prenylated derivatives were identified as minor products. In comparison to the known tryptophan-containing cyclic dipeptide C7-prenyltransferase CTrpPT from Aspergillus oryzae, CdpC7PT showed a much higher substrate flexibility. It also accepted cyclo-l-Tyr-l-Tyr as substrate and catalyzed an O-prenylation at the tyrosyl residue, providing the first example from the dimethylallyltryptophan synthase (DMATS) superfamily with an O-prenyltransferase activity towards dipeptides. Furthermore, products with both C7-prenyl at tryptophanyl and O-prenyl at tyrosyl residue were detected in the reaction mixture of cyclo-l-Trp-l-Tyr. Determination of the kinetic parameters proved that (S)-benzodiazepinedione consisting of a tryptophanyl and an anthranilyl moiety was accepted as the best substrate with a K _M value of 204.1 μM and a turnover number of 0.125 s^?1. Cyclo-l-Tyr-l-Tyr was accepted with a K _M value of 1,411.3 μM and a turnover number of 0.012 s^?1.     

Efficient production of indigoidine in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Indigoidine is a bacterial natural product with antioxidant and antimicrobial activities. Its bright blue color resembles the industrial dye indigo, thus representing a new natural blue dye that may find uses in industry. In our previous study, an indigoidine synthetase Sc-IndC and an associated helper protein Sc-IndB were identified from Streptomyces chromofuscus ATCC 49982 and successfully expressed in Escherichia coli BAP1 to produce the blue pigment at 3.93 g/l. To further improve the production of indigoidine, in this work, the direct biosynthetic precursor l-glutamine was fed into the fermentation broth of the engineered E. coli strain harboring Sc-IndC and Sc-IndB. The highest titer of indigoidine reached 8.81 ± 0.21 g/l at 1.46 g/l l-glutamine. Given the relatively high price of l-glutamine, a metabolic engineering technique was used to directly enhance the in situ supply of this precursor. A glutamine synthetase gene (glnA) was amplified from E. coli and co-expressed with Sc-indC and Sc-indB in E. coli BAP1, leading to the production of indigoidine at 5.75 ± 0.09 g/l. Because a nitrogen source is required for amino acid biosynthesis, we then tested the effect of different nitrogen-containing salts on the supply of l-glutamine and subsequent indigoidine production. Among the four tested salts including (NH₄)₂SO₄, NH₄Cl, (NH₄)₂HPO₄ and KNO₃, (NH₄)₂HPO₄ showed the best effect on improving the titer of indigoidine. Different concentrations of (NH₄)₂HPO₄ were added to the fermentation broths of E. coli BAP1/Sc-IndC+Sc-IndB+GlnA, and the titer reached the highest (7.08 ± 0.11 g/l) at 2.5 mM (NH₄)₂HPO₄. This work provides two efficient methods for the production of this promising blue pigment in E. coli.     

Engineering rTCA pathway and C4-dicarboxylate transporter for <Emphasis Type="SmallCaps">l</Emphasis>-malic acid production

l-Malic acid is an important component of a vast array of food additives, antioxidants, disincrustants, pharmaceuticals, and cosmetics. Here, we presented a pathway optimization strategy and a transporter modification approach to reconstruct the l-malic acid biosynthesis pathway and transport system, respectively. First, pyruvate carboxylase (pyc) and malate dehydrogenase (mdh) from Aspergillus flavus and Rhizopus oryzae were combinatorially overexpressed to construct the reductive tricarboxylic acid (rTCA) pathway for l-malic acid biosynthesis. Second, the l-malic acid transporter (Spmae) from Schizosaccharomyces pombe was engineered by removing the ubiquitination motification to enhance the l-malic acid efflux system. Finally, the l-malic acid pathway was optimized by controlling gene expression levels, and the final l-malic acid concentration, yield, and productivity were up to 30.25 g L^?1, 0.30 g g^?1, and 0.32 g L^?1 h^?1 in the resulting strain W4209 with CaCO₃ as a neutralizing agent, respectively. In addition, these corresponding parameters of pyruvic acid remained at 30.75 g L^?1, 0.31 g g^?1, and 0.32 g L^?1 h^?1, respectively. The metabolic engineering strategy used here will be useful for efficient production of l-malic acid and other chemicals.     

The modulation of NMDA receptors and <Emphasis Type="SmallCaps">l</Emphasis>-arginine/nitric oxide pathway is implicated in the anti-immobility effect of creatine in the tail suspension test

The modulation of N-methyl-D-aspartate receptor (NMDAR) and l-arginine/nitric oxide (NO) pathway is a therapeutic strategy for treating depression and neurologic disorders that involves excitotoxicity. Literature data have reported that creatine exhibits antidepressant and neuroprotective effects, but the implication of NMDAR and l-arginine/nitric oxide (NO) pathway in these effects is not established. This study evaluated the influence of pharmacological agents that modulate NMDAR/l-arginine-NO pathway in the anti-immobility effect of creatine in the tail suspension test (TST) in mice. The NOx levels and cellular viability in hippocampal and cerebrocortical slices of creatine-treated mice were also evaluated. The anti-immobility effect of creatine (10 mg/kg, po) in the TST was abolished by NMDA (0.1 pmol/mouse, icv), d-serine (30 µg/mouse, icv, glycine-site NMDAR agonist), arcaine (1 mg/kg, ip, polyamine site NMDAR antagonist), l-arginine (750 mg/kg, ip, NO precursor), SNAP (25 μg/mouse, icv, NO donor), L-NAME (175 mg/kg, ip, non-selective NOS inhibitor) or 7-nitroindazole (50 mg/kg, ip, neuronal NOS inhibitor), but not by DNQX (2.5 µg/mouse, icv, AMPA receptor antagonist). The combined administration of sub-effective doses of creatine (0.01 mg/kg, po) and NMDAR antagonists MK-801 (0.001 mg/kg, po) or ketamine (0.1 mg/kg, ip) reduced immobility time in the TST. Creatine (10 mg/kg, po) increased cellular viability in hippocampal and cerebrocortical slices and enhanced hippocampal and cerebrocortical NO _x levels, an effect potentiated by l-arginine or SNAP and abolished by 7-nitroindazole or L-NAME. In conclusion, the anti-immobility effect of creatine in the TST involves NMDAR inhibition and enhancement of NO levels accompanied by an increase in neural viability.     

Characterization of an α-amino-ɛ-caprolactam racemase with broad substrate specificity from <Emphasis Type="Italic">Citreicella</Emphasis> sp. SE45

α-Amino-ε-caprolactam (ACL) racemizing activity was detected in a putative dialkylglycine decarboxylase (EC 4.1.1.64) from Citreicella sp. SE45. The encoding gene of the enzyme was cloned and transformed in Escherichia coli BL21 (DE3). The molecular mass of the enzyme was shown to be 47.4 kDa on SDS–polyacrylamide gel electrophoresis. The enzymatic properties including pH and thermal optimum and stabilities were determined. This enzyme acted on a broad range of amino acid amides, particularly unbranched amino acid amides including l-alanine amide and l-serine amide with a specific activity of 17.5 and 21.6 U/mg, respectively. The K _m and V _max values for d- and l-ACL were 5.3 and 2.17 mM, and 769 and 558 μmol/min.mg protein, respectively. Moreover, the turn over number (K _cat) and catalytic efficiency (K _cat/K _m ) of purified ACL racemase from Citreicella sp. SE45 using l-ACL as a substrate were 465 S^?1 and 214 S^?1mM^?1, respectively. The new ACL racemase from Citreicella sp. SE45 has a potential to be used as the biocatalytic application.     

Ancient split of major genetic lineages of European Black Pine: evidence from chloroplast <Emphasis>DNA</Emphasis>

The European Black Pine (Pinus nigra Arn.) has a long and complex history. Genetic distance and frequency analyses identified three differentiated genetic groups, which corresponded to three wide geographical areas: Westerns Mediterranean, Balkan Peninsula and Asia Minor. These groups shared common ancestors (14.75 and 10.72 Ma). The most recent splits occurred after the Messinian Salinity Crisis (4.37 Ma) and the Early–Middle Pleistocene Transitions (0.93 Ma). The posterior ancestral population size (Na) is 260,000–265,000 individuals. Each pool is further fragmented, with evidence of a phylogeographic structure (N _st  > G _st ) typically observed in some natural populations from the Western Mediterranean region and the Balkan Peninsula. The laboratory analysis was performed by fragment analysis—i.e. electrophoretic sizing of polymerase chain reaction fragments, combined with the sequencing analysis of 33 % of all individuals as a control. Intense sampling of chloroplast DNA polymorphisms (3154 individuals and 13 markers: SNPs and SSRs) over the full area of the species’ natural distribution indicated moderate among-population variability (G _st(nc) ≤ 0.177) in various parts of its range. These results indicate that the natural populations have long migration histories that differ from one another and that they have been strongly phylogeographically affected by complex patterns of isolation, speciation and fragmentation. Long and varying climatic fluctuations in the region of the principal genetic group have been the probable cause of different forest community associations with different successional patterns resulting in interglacial refugia vs. macro long-term refugia.     

Genetic assessment of <Emphasis Type="Italic">Abies koreana</Emphasis> (Pinaceae), the endangered Korean fir,and conservation implications

To establish a management plan for endangered and rare species, genetic assessment must first be conducted. The genetic characteristics of plant species are affected by demographic history, reproductive strategy, and distributional range as well as anthropological effects. Abies koreana E. H. Wilson (Pinaceae), Korean fir, is endemic to Korea and found only in sub-alpine areas of the southern Korean Peninsula and Jejudo Island. This species has been designated as critically endangered by the International Union for Conservation of Nature due to a continuous decline in its range and population fragmentation. We genotyped 176 individuals from seven natural populations and two afforested populations on the Korean Peninsula using 19 microsatellite loci. STRUCTURE analysis revealed two genetic clusters in natural populations (F _st  = 0.040 and R _st  = 0.040) despite low differentiation. We did not detect a significant reduction in genetic diversity or the signature of a genetic bottleneck despite population fragmentation and small population size. We deduced that this species exhibits a metapopulation structure, with the population on Jirisan Mountain acting as a source of genetic diversity for other local small populations on the Korean Peninsula, through contemporary asymmetric gene flow. However, the majority of afforested individuals on the Korean Peninsula originated from a different gene cluster. Thus, we recommend a conservation strategy that maintains two genetically unique clusters.     

Genetic differentiation among natural populations of the lizard complex <Emphasis Type="Italic">Darevskia raddei</Emphasis> as inferred from genome microsatellite marking

The article presents the genetic parameters of the populations of lizards of the Darevskia raddei complex (D. raddei nairensis and D. raddei raddei) and the populations of D. valentini calculated on the basis of the analysis of variability of 50 allelic variants of the three nuclear genome microsatellite-containing loci of 83 individuals. It was demonstrated that the F_st genetic distances between the populations of D. raddei nairensis and D. raddei raddei were not statistically significantly different from the F_st genetic distances between the populations of different species, D. raddei and D. valentini. At the same time, these distances were statistically significantly higher than the F_st distances between the populations belonging to one species within the genus Darevskia. These data suggest deep divergence between the populations of D. raddei raddei and D. raddei nairensis of the D. raddei complex and there arises the question on considering them as separate species.     

Characterization of aspartate kinase and homoserine dehydrogenase from <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> IWJ001 and systematic investigation of <Emphasis Type="SmallCaps">l</Emphasis>-isoleucine biosynthesis

Previously we have characterized a threonine dehydratase mutant TD^F383V (encoded by ilvA1) and an acetohydroxy acid synthase mutant AHAS^{P176S, D426E, L575W} (encoded by ilvBN1) in Corynebacterium glutamicum IWJ001, one of the best l-isoleucine producing strains. Here, we further characterized an aspartate kinase mutant AK^A279T (encoded by lysC1) and a homoserine dehydrogenase mutant HD^G378S (encoded by hom1) in IWJ001, and analyzed the consequences of all these mutant enzymes on amino acids production in the wild type background. In vitro enzyme tests confirmed that AK^A279T is completely resistant to feed-back inhibition by l-threonine and l-lysine, and that HD^G378S is partially resistant to l-threonine with the half maximal inhibitory concentration between 12 and 14 mM. In C. glutamicum ATCC13869, expressing lysC1 alone led to exclusive l-lysine accumulation, co-expressing hom1 and thrB1 with lysC1 shifted partial carbon flux from l-lysine (decreased by 50.1 %) to l-threonine (4.85 g/L) with minor l-isoleucine and no l-homoserine accumulation, further co-expressing ilvA1 completely depleted l-threonine and strongly shifted carbon flux from l-lysine (decreased by 83.0 %) to l-isoleucine (3.53 g/L). The results demonstrated the strongly feed-back resistant TD^F383V might be the main driving force for l-isoleucine over-synthesis in this case, and the partially feed-back resistant HD^G378S might prevent the accumulation of toxic intermediates. Information exploited from such mutation-bred production strain would be useful for metabolic engineering.     

Cytotoxicity and Glycan-Binding Profile of a <Emphasis Type="SmallCaps">d</Emphasis>-Galactose-Binding Lectin from the Eggs of a Japanese Sea Hare (<Emphasis Type="Italic">Aplysia kurodai</Emphasis>)

A divalent cation-independent 16 kDa d-galactose binding lectin (AKL-2) was isolated from eggs of sea hare, Aplysia kurodai. The lectin recognized d-galactose and d-galacturonic acid and had a 32 kDa dimer consisting of two disulfide-bonded 16 kDa subunits. Eighteen N-terminus amino acids were identified by Edman degradation, having unique primary structure. Lectin blotting analysis with horseradish peroxidase-conjugated lectins has shown that AKL-2 was a glycoprotein with complex type oligosaccharides with N-acetyl d-glucosamine and mannose at non-reducing terminal. Two protein bands with 38 and 36 kDa in the crude extract of sea hare eggs after purification of the lectin was isolated by AKL-2-conjugated Sepharose column and elution with 0.1 M lactose containing buffer. It suggested that the lectin binds with an endogenous ligand in the eggs. AKL-2 kept extreme stability on haemagglutination activity if it was treated at pH 3 and 70 °C for 1 h. Glycan binding profile of AKL-2 by frontal affinity chromatography technology using 15 pyridylamine labeled oligosaccharides has been appeared that the lectin uniquely recognized globotriose (Galα1-4Galβ1-4Glc; Gb3) in addition to bi-antennary complex type N-linked oligosaccharides with N-acetyllactosamine. Surface plasmon resonance analysis of AKL-2 against a neo-glycoprotein, Gb3-human serum albumin showed the k _ass and k _diss values are 2.4 × 10³ M^?1 s^?1 and 3.8 × 10^?3 s^?1, respectively. AKL-2 appeared cytotoxicity against both Burkitt’s lymphoma Raji cell and erythroleukemia K562. The activity to Raji by the lectin was preferably cancelled by the co-presence of melibiose mimicing Gb3. On the other hand, K562 was cancelled effectively by lactose than melibiose. It elucidated that AKL-2 had cytotoxic ability mediated glycans structure to cultured cells.     

Isolation and characterization of a novel <Emphasis Type="SmallCaps">l</Emphasis>-glutamate oxidase with strict substrate specificity from <Emphasis Type="Italic">Streptomyces diastatochromogenes</Emphasis>

Objectives

To find an l-glutamate oxidase (LGox), to be used for the quantitative analysis of l-glutamic acid, an lgox gene encoding LGox from Streptomyces diastatochromogenes was isolated, cloned and characterized.

Results

The gene had an ORF of 1974 bp encoding a protein of 657 amino acid residues. In comparison to the LGox precursor, the proteinase K-treated enzyme exhibited improved affinity to substrate and with a K _m of 0.15 mM and V _max of 62 μmol min^?1 mg^?1. The 50% thermal inactivation temperature of the proteinase K treated enzyme was increased from 50 to 70 °C. The enzyme exhibited strict specificity for l-glutamate.

Conclusions

LGox treated by proteinase K exhibited strict specificity for l-glutamate, good thermostability and high substrate affinity.

     

A saponin isolated from <Emphasis Type="Italic">Agapanthus africanus</Emphasis> differentially induces apoplastic peroxidase activity in wheat and displays fungicidal properties

A spirostane with an attached trisaccharide, (25R)-5α-spirostane-2α,3β,5α-triol 3-O-(O-α-l-rhamnopyranosyl-(1 → 2)-O-(β-d-galactopyranosyl-(1 → 3))-β-d-glucopyranoside), was isolated and identified from the aerial parts of Agapanthus africanus by activity-guided fractionation. Fungicidal properties of the crude extract, semi-purified fractions as well as the purified active saponin from A. africanus were screened in vitro against Fusarium oxysporum. At a concentration of 1 mg mL^?1, the crude extract and semi-purified ethyl acetate and dichloromethane fractions showed significant antifungal activity. The purified saponin inhibited the in vitro mycelial growth of F. oxysporum completely (100 %) at a concentration of 125 µg mL^?1. Furthermore, to verify previously observed induced resistance by crude extracts of A. africanus towards leaf rust, intercellular PR-protein activity was determined in wheat seedlings following foliar application of the purified saponin at 100 µg mL^?1. In vitro peroxidase enzyme activity increased significantly (60 %) in wheat seedlings 48 h after treatment with the purified saponin, demonstrating its role as an elicitor to activate a defence reaction in wheat.     

Sap flow of the southern conifer, <Emphasis Type="Italic">Agathis australis</Emphasis> during wet and dry summers

Key message

Analysis of sap flux density during drought suggests that the large sapwood and rooting volumes of larger trees provide a buffer against drying soil.

Abstract

The southern conifer Agathis australis is amongst the largest and longest-lived trees in the world. We measured sap flux densities (F _d) in kauri trees with a DBH range of 20–176 cm to explore differences in responses of trees of different sizes to seasonal conditions and summer drought. F _d was consistently higher in larger trees than smaller trees. Peak F _d was 20 and 8 g m^?2 s^?1 for trees of diameters of 176 and 20 cm, respectively, during the wet summer. Multiple regression analysis revealed photosynthetically active radiation (PAR) and vapour pressure deficit (D) were the main drivers of F _d. During drought, larger trees were more responsive to D whilst smaller trees were more responsive to soil drying. Our largest tree had a sapwood area of 3,600 cm². Preliminary analysis suggests stem water storage provides a buffer against drying soil in larger trees. Furthermore, F _d of smaller trees had higher R ² values for soil moisture at 30 and 60 cm depth than soil moisture at 10 cm depth (R ² = 0.68–0.97 and 0.55–0.67, respectively) suggesting that deeper soil moisture is more important for these trees. Larger trees did not show a relationship between F _d and soil moisture, suggesting they were accessing soil water deeper than 60 cm. These results suggest that larger trees may be better prepared for increasing frequency and intensity of summer droughts due to deeper roots and/or larger stem water storage capacity.

     

Investigating the genetic diversity and differentiation patterns in the <Emphasis Type="Italic">Penstemon scariosus</Emphasis> species complex under different sample sizes using AFLPs and SSRs

Habitat fragmentation due to anthropogenic activities is the major cause of biodiversity loss. Endemic and narrowly distributed species are the most susceptible to habitat degradation. Penstemon scariosus is one of many species whose natural habitat is vulnerable to industrialization. All varieties of P. scariosus (P. scariosus var. albifluvis, P. scariosus var. cyanomontanus, P. scariosus var. garrettii, P. scariosus var. scariosus) have small distribution ranges, but only P. scariosus var. albifluvis is being considered for listing under the Endangered Species Act. We used eight microsatellites or simple sequence repeats (SSRs) loci and two amplified fragment length polymorphism (AFLP) primer combinations to investigate the population genetic structure and diversity of P. scariosus varieties. Moreover, we compared the utility of the two marker systems in conservation genetics and estimated an appropriate sample size in population genetic studies. Genetic differentiation among populations based on F_st ranged from low to moderate (F_st?=?0.056–0.157) and from moderate to high when estimated with D_es (D_es?=?0.15–0.32). Also, AMOVA analysis shows that most of the genetic variation is within populations. Inbreeding coefficients (F_is) were high in all varieties (0.20–0.56). The Bayesian analysis, STRUCTURE, identified three clusters from SSR data and four clusters from AFLPs. Clusters were not consistent between marker systems and did not represent the current taxonomy. MEMGENE revealed that a high proportion of the genetic variation is due to geographic distance (R²?=?0.38, P?=?0.001). Comparing the genetic measurements from AFLPs and SSRs, we found that AFLP results were more accurate than SSR results across sample size when populations were larger than 25 individuals. As sample size decreases, the estimates become less stable in both AFLP and SSR datasets. Finally, this study provides insight into the population genetic structure of these varieties, which could be used in conservation efforts.     

Rangewide genetic analysis of Lesser Prairie-Chicken reveals population structure,range expansion,and possible introgression

The distribution of the Lesser Prairie-Chicken (Tympanuchus pallidicinctus) has been markedly reduced due to loss and fragmentation of habitat. Portions of the historical range, however, have been recolonized and even expanded due to planting of conservation reserve program (CRP) fields that provide favorable vegetation structure for Lesser Prairie-Chickens. The source population(s) feeding the range expansion is unknown, yet has resulted in overlap between Lesser and Greater Prairie-Chickens (T. cupido) increasing the potential for hybridization. Our objectives were to characterize connectivity and genetic diversity among populations, identify source population(s) of recent range expansion, and examine hybridization with the Greater Prairie-Chicken. We analyzed 640 samples from across the range using 13 microsatellites. We identified three to four populations corresponding largely to ecoregions. The Shinnery Oak Prairie and Sand Sagebrush Prairie represented genetically distinct populations (F _ST > 0.034 and F _ST > 0.023 respectively). The Shortgrass/CRP Mosaic and Mixed Grass ecoregions appeared admixed (F _ST = 0.009). Genetic diversity was similar among ecoregions and N _e ranged from 142 (95 % CI 99–236) for the Shortgrass/CRP Mosaic to 296 (95 % CI 233–396) in the Mixed Grass Prairie. No recent migration was detected among ecoregions, except asymmetric dispersal from both the Mixed Grass Prairie and to a lesser extent the Sand Sagebrush Prairie north into adjacent Shortgrass/CRP Mosaic (m = 0.207, 95 % CI 0.116–0.298, m = 0.097, 95 % CI 0.010–0.183, respectively). Indices investigating potential hybridization in the Shortgrass/CRP Mosaic revealed that six of the 13 individuals with hybrid phenotypes were significantly admixed suggesting hybridization. Continued monitoring of diversity within and among ecoregions is warranted as are actions promoting genetic connectivity and range expansion.     

Enhancement of medium-chain-length polyhydroxyalkanoates biosynthesis from glucose by metabolic engineering in <Emphasis Type="Italic">Pseudomonas mendocina</Emphasis>

Objectives

To enhance the biosynthesis of medium-chain-length polyhydroxyalkanoates (PHA_MCL) from glucose in Pseudomonas mendocina NK-01, metabolic engineering strategies were used to block or enhance related pathways.

Results

Pseudomonas mendocina NK-01 produces PHA_MCL from glucose. Besides the alginate oligosaccharide biosynthetic pathway proved by our previous study, UDP-d-glucose and dTDP-l-rhamnose biosynthetic pathways were identified. These might compete for glucose with the PHA_MCL biosynthesis. First, the alg operon, galU and rmlC gene were deleted one by one, resulting in NK-U-1(?alg), NK-U-2 (?alg?galU), NK-U-3(alg?galU?rmlC). After fermentation for 36 h, the cell dry weight (CDW) and PHA_MCL production of these strains were determined. Compared with NK-U: 1) NK-U-1 produced elevated CDW (from 3.19 ± 0.16 to 3.5 ± 0.11 g/l) and equal PHA_MCL (from 0.78 ± 0.06 to 0.79 ± 0.07 g/l); 2) NK-U-2 produced more CDW (from 3.19 ± 0.16 to 3.55 ± 0.23 g/l) and PHA_MCL (from 0.78 ± 0.06 to 1.05 ± 0.07 g/l); 3) CDW and PHA_MCL dramatically decreased in NK-U-3 (1.53 ± 0.21 and 0.41 ± 0.09 g/l, respectively). Additionally, the phaG gene was overexpressed in strain NK-U-2. Although CDW of NK-U-2/phaG decreased to 1.29 ± 0.2 g/l, PHA titer (%CDW) significantly increased from 24.5 % up to 51.2 %.

Conclusion

The PHA_MCL biosynthetic pathway was enhanced by blocking branched metabolic pathways in combination with overexpressing phaG gene.

     

Unspecific histological and hematological alterations in anadromous and resident <Emphasis Type="Italic">Salvelinus malma</Emphasis> induced by volcanogenic pollution

Knowledge of larval dispersal and connectivity in coral reef species is crucial for understanding population dynamics, resilience, and evolution of species. Here, we use ten microsatellites and one mitochondrial marker (cytochrome b) to investigate the genetic population structure, genetic diversity, and historical demography of the powder-blue tang Acanthurus leucosternon across more than 1000 km of the scarcely studied Eastern African region. The global AMOVA results based on microsatellites reveal a low but significant F _ST value (F _ST = 0.00252 P < 0.001; D _EST = 0.025 P = 0.0018) for the 336 specimens sampled at ten sample sites, while no significant differentiation could be found in the mitochondrial cytochrome b dataset. On the other hand, pairwise F _ST, PCOA, and hierarchical analysis failed to identify any genetic breaks among the Eastern African populations, supporting the hypothesis of genetic homogeneity. The observed genetic homogeneity among Eastern African sample sites can be explained by the lengthy post-larval stage of A. leucosternon, which can potentiate long-distance dispersal. Tests of neutrality and mismatch distribution signal a population expansion during the mid-Pleistocene period.