Phosphoenolpyruvate-supply module in <Emphasis Type="Italic">Escherichia coli</Emphasis> improves <Emphasis Type="Italic">N</Emphasis>-acetyl-<Emphasis Type="SmallCaps">d</Emphasis>-neuraminic acid biocatalysis

Objectives

N-Acetyl-d-neuraminic acid (Neu5Ac) is often synthesized from exogenous N-acetylglucosamine (GlcNAc) and excess pyruvate. We have previously constructed a recombinant Escherichia coli strain for Neu5Ac production using GlcNAc and intracellular phosphoenolpyruvate (PEP) as substrates (Zhu et al. Biotechnol Lett 38:1–9, 2016).

Results

PEP synthesis-related genes, pck and ppsA, were overexpressed within different modes to construct PEP-supply modules, and their effects on Neu5Ac production were investigated. All the PEP-supply modules enhanced Neu5Ac production. For the best module, pCDF-pck-ppsA increased Neu5Ac production to 8.6 ± 0.15 g l^?1, compared with 3.6 ± 0.15 g l^?1 of the original strain. Neu5Ac production was further increased to 15 ± 0.33 g l^?1 in a 1 l fermenter.

Conclusions

The PEP-supply module can improve the intracellular PEP supply and enhance Neu5Ac production, which benefited industrial Neu5Ac production.

     

Production of <Emphasis Type="Italic">trans</Emphasis>-10,<Emphasis Type="Italic">cis</Emphasis>-12-conjugated linoleic acid using permeabilized whole-cell biocatalyst of <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

Objective

To improve the production of trans-10,cis-12-conjugated linoleic acid (t10,c12-CLA) from linoleic acid in recombinant Yarrowia lipolytica.

Results

Cells of the yeast were permeabilized by freeze/thawing. The optimal conditions for t10,c12-CLA production by the permeabilized cells were at 28 °C, pH 7, 200 rpm with 1.5 g sodium acetate l^?1, 100 g wet cells l^?1, and 25 g LA l^?1. Under these conditions, the permeabilized cells produced 15.6 g t10,c12-CLA l^?1 after 40 h, with a conversion yield of 62 %. The permeabilized cells could be used repeatedly for three cycles, with the t10,c12-CLA extracellular production remaining above 10 g l^?1.

Conclusion

Synthesis of t10,c12-CLA was achieved using a novel method, and the production reported in this work is the highest value reported to date.

     

Productivity and biochemical composition of <Emphasis Type="Italic">Tetradesmus obliquus</Emphasis> and <Emphasis Type="Italic">Phaeodactylum tricornutum</Emphasis>: effects of different cultivation approaches

The present work evaluated biomass productivity, carbon dioxide fixation rate, and biochemical composition of two microalgal species, Phaeodactylum tricornutum (Bacillariophyta) and Tetradesmus obliquus (Chlorophyta), cultivated indoors in high-technology photobioreactors (HT-PBR) and outdoors both in pilot ponds and low-technology photobioreactors in a greenhouse in southern Italy. Microalgae were grown in standard media, under nitrogen starvation, and in two liquid digestates obtained from anaerobic digestion of agro-zootechnical and vegetable biomass. P. tricornutum, cultivated in semi-continuous mode in indoor HT-PBRs with standard medium, showed a biomass productivity of 21.0?±?2.3 g m^?2 d^?1. Applying nitrogen starvation, the lipid productivity increased from 2.3 up to 4.5?±?0.5 g m^?2 d^?1, with a 24 % decrease of biomass productivity. For T. obliquus, a biomass productivity of 9.1?±?0.9 g m^?2 d^?1 in indoor HT-PBR was obtained using standard medium. Applying liquid digestates as fertilizers in open ponds, T. obliquus gave a biomass productivity (10.8?±?2.0 g m^?2 d^?1) not statistically different from complete medium such as P. tricornutum (6.5?±?2.2 g m^?2 d^?1). The biochemical data showed that the fatty acid composition of the microalgal biomass was affected by the different cultivation conditions for both microalgae. In conclusion, it was found that the microalgal productivity in standard medium was about doubled in HT-PBR compared to open ponds for P. tricornutum and was about 20 % higher for T. obliquus.     

<Emphasis Type="Italic">Nibribacter flagellatus</Emphasis> sp. nov., isolated from rhizosphere of <Emphasis Type="Italic">Hibiscus syriacus</Emphasis> and emended description of the genus <Emphasis Type="Italic">Nibribacter</Emphasis>

A Gram-stain negative, aerobic, motile by flagella, rod-shaped strain (THG-T16^T) was isolated from rhizosphere of Hibiscus syriacus. Growth occurred at 10–40 °C (optimum 28–30 °C), at pH 6.0–8.0 (optimum 7.0) and at 0–1.0% NaCl (optimum 0%). Based on 16S rRNA gene sequence analysis, the near phylogenetic neighbours of strain THG-T16^T were identified as Nibribacter koreensis KACC 16450^T (98.6%), Rufibacter roseus KCTC 42217^T (94.7%), Rufibacter immobilis CCTCC AB 2013351^T (94.5%) and Rufibacter tibetensis CCTCC AB 208084^T (94.4%). The DNA G+C content of strain THG-T16^T was determined to be 46.7 mol%. DNA–DNA hybridization values between strain THG-T16^T and N. koreensis KACC 16450^T, R. roseus KCTC 42217^T, R. immobilis CCTCC AB 2013351^T, R.tibetensis CCTCC AB 208084^T were 33.5?±?0.5% (31.7?±?0.7% reciprocal analysis), 28.1?±?0.2% (25.2?±?0.2%), 17.1?±?0.9% (10.2?±?0.6%) and 8.1?±?0.3% (5.2?±?0.1%). The polar lipids were identified as phosphatidylethanolamine, two unidentified aminophospholipids, an unidentified aminolipid and three unidentified lipids. The quinone was identified as MK-7 and the polyamine as sym-homospermidine. The major fatty acids were identified as C_16:1 ω5c, C_17:1 ω6c, iso-C_15:0, summed feature 3 (C_16:1 ω7c and/or C_16:1 ω6c) and summed feature 4 (iso-C_17:1 I and/or anteiso-C_17:1 B). On the basis of the phylogenetic analysis, chemotaxonomic data, physiological characteristics, and DNA–DNA hybridization data, strain THG-T16^T represents a novel species of the genus Nibribacter, for which the name Nibribacter flagellatus sp. nov. is proposed. The type strain is THG-T16^T(=?KACC 19188^T?=?CCTCC AB 2016246^T).     

Effect of over-expression of <Emphasis Type="Italic">Linum usitatissimum</Emphasis> PINORESINOL LARICIRESINOL REDUCTASE (<Emphasis Type="Italic">LuPLR</Emphasis>) gene in transgenic <Emphasis Type="Italic">Phyllanthus amarus</Emphasis>

Shoot tip explants of Phyllanthus amarus were cocultivated with Agrobacterium tumefaciens strain LBA 4404 carrying plasmid pCAMBIA 2301 harbouring genes coding for betaglucuronidase (gus), kanamycin (kan), and neomycin phosphotransferase II (nptII) along with a gene coding for Linum usitatissimum PINORESINOL LARICIRESINOL REDUCTASE (Lu-PLR). Transformed shoot tip explants were maintained in a Murashige and Skoog (MS) medium containing TDZ 1.54 mg l^?1, kan 50 mg l^?1 and cephotaxime 62.5 mg l^?1. The optimum medium for regeneration of multiple shoots was MS supplemented with TDZ 1.54 mg l^?1, kan 50 mg l^?1. Efficient and effective rooting of plantlets was achieved by culturing the in vitro regenerated shoots on liquid ½ MS medium containing 0.7 mg l^?1 indole 3-butyric acid (IBA) and 5 mg l^?1 kan. Rooted plants were acclimatized in the mixtures of vermiculite and soil. The transformation of kan-resistant plantlets regenerated from shoot-tip explants was confirmed by GUS and polymerase chain reaction (PCR) analysis. Southern blot and reverse transcribed PCR (RT-PCR) analysis confirmed successful integration and expression of Lu-PLR gene. Quantitative analysis of phyllanthin performed on transgenic and wild plants using high-performance liquid chromatography (HPLC) revealed that transgenic lines contained higher phyllanthin content (0.3–0.81% w/w) than wild plants (0.09% w/w). The highest yield of phyllanthin was detected in transgenic lines was up to 1.16, 1.22 and 1.23 folds higher than that of wild plant. This report highlights the transgenic approach to enhance the contents of phyllanthin and hypophyllanthin.     

Feeding capability of black scraper <Emphasis Type="Italic">Thamnaconus modestus</Emphasis> on giant jellyfish <Emphasis Type="Italic">Nemopilema nomurai</Emphasis> evaluated through field observations and tank experiments

Although many fish species are known to feed on jellyfish, only a few quantitative studies on this subject have been conducted in the field or laboratory. We combined field observations and feeding experiments to quantitatively evaluate the potential feeding capability of the black scraper Thamnaconus modestus on giant jellyfish Nemopilema nomurai in a jellyfish-abundant area. Underwater observations revealed that shoals of T. modestus fed voraciously on jellyfish when the jellyfish were close to the fish’s seaweed habitat. Gut contents of field-collected T. modestus almost exclusively consisted of jellyfish. In tank trials, feeding activity of black scraper was high when light intensity was between about 1.1 lx and 50 × 10³ lx. The estimated feeding speed of T. modestus on the jellyfish in the Sea of Japan when the jellyfish bloom occurred was 10.0 ± 2.0 times fish body weight per day. The results support the prediction that T. modestus and probably other coastal medusivorous fishes have a substantial capability to eliminate jellyfish blooms. Considering that they are highly dependent on seaweed bed, protection of such habitats for these medusivores may be the most cost-efficient control method for jellyfish blooms.     

The diversity of rhizobia nodulating the <Emphasis Type="Italic">Medicago</Emphasis>, <Emphasis Type="Italic">Melilotus</Emphasis> and <Emphasis Type="Italic">Trigonella</Emphasis> inoculation group in Egypt is marked by the dominance of two genetic types

Twenty four rhizobial strains were isolated from root nodules of Melilotus, Medicago and Trigonella plants growing wild in soils throughout Egypt. The nearly complete 16S rRNA gene sequence from each strain showed that 12 strains (50 %) were closely related to the Ensifer meliloti LMG6133^T type strain with identity values higher than 99.0 %, that 9 (37.5 %) strains were more than 99 % identical to the E. medicae WSM419^T type strain, and that 3 (12.5 %) strains showed 100 % identity with the type strain of N. huautlense S02^T. Accordingly, the diversity of rhizobial strains nodulating wild Melilotus, Medicago and Trigonella species in Egypt is marked by predominance of two genetic types, E. meliloti and E. medicae, although the frequency of isolation was slightly higher in E. meliloti. Sequencing of the symbiotic nodC gene from selected Medicago and Melilotus strains revealed that they were all similar to those of the E. meliloti LMG6133^T and E. medicae WSM419^T type strains, respectively. Similarly, nodC sequences of strains identified as members of the genus Neorhizobium were more than 99 % identical to that of N. galegae symbiovar officinalis HAMBI 114.     

Raw starch conversion by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> expressing <Emphasis Type="Italic">Aspergillus tubingensis</Emphasis> amylases

Background

Starch is one of the most abundant organic polysaccharides available for the production of bio-ethanol as an alternative transport fuel. Cost-effective utilisation of starch requires consolidated bioprocessing (CBP) where a single microorganism can produce the enzymes required for hydrolysis of starch, and also convert the glucose monomers to ethanol.

Results

The Aspergillus tubingensis T8.4 α-amylase (amyA) and glucoamylase (glaA) genes were cloned and expressed in the laboratory strain Saccharomyces cerevisiae Y294 and the semi-industrial strain, S. cerevisiae Mnuα1. The recombinant AmyA and GlaA displayed protein sizes of 110–150 kDa and 90 kDa, respectively, suggesting significant glycosylation in S. cerevisiae. The Mnuα1[AmyA-GlaA] and Y294[AmyA-GlaA] strains were able to utilise 20 g l^-1 raw corn starch as sole carbohydrate source, with ethanol titers of 9.03 and 6.67 g l^-1 (0.038 and 0.028 g l^-1 h^-1), respectively, after 10 days. With a substrate load of 200 g l^-1 raw corn starch, Mnuα1[AmyA-GlaA] yielded 70.07 g l^-1 ethanol (0.58 g l^-1 h^-1) after 120 h of fermentation, whereas Y294[AmyA-GlaA] was less efficient at 43.33 g l^-1 ethanol (0.36 g l^-1 h^-1).

Conclusions

In a semi-industrial amylolytic S. cerevisiae strain expressing the A. tubingensis α-amylase and glucoamylase genes, 200 g l^-1 raw starch was completely hydrolysed (saccharified) in 120 hours with 74% converted to released sugars plus fermentation products and the remainder presumably to biomass. The single-step conversion of raw starch represents significant progress towards the realisation of CBP without the need for any heat pretreatment. Furthermore, the amylases were produced and secreted by the host strain, thus circumventing the need for exogenous amylases.

     

Linkages of <Emphasis Type="Italic">Firmicutes</Emphasis> and <Emphasis Type="Italic">Bacteroidetes</Emphasis> populations to methanogenic process performance

To identify potential linkages between specific bacterial populations and process performance in anaerobic digestion, the dynamics of bacterial community structure was monitored with high-throughput sequencing in triplicate anaerobic digesters treating animal waste. Firmicutes and Bacteroidetes were found as the two most abundant populations, however, with contrasting population dynamics in response to organic overloading. Firmicutes dominated the bacterial community during stable process performance at low organic loading rate, representing over 50 % of the bacterial abundance. In contrast, the onset of organic overloading raised the relative abundance of Bacteroidetes from 20 ± 2.6 to 44 ± 3.1 %. In addition to the significant negative correlation between the relative abundance of Firmicutes and Bacteroidetes, populations of Firmicutes and Bacteroidetes were found to be linked to process parameters including organic loading rate, volatile fatty acids concentration, and methane production. Therefore, the population abundance ratio of Firmicutes to Bacteroidetes (F/B ratio) was suggested as a potential indicator for process performance. The interactions between Firmicutes and Bacteroidetes populations could be exploited to develop strategies for the prevention of performance perturbation in anaerobic digestion processes.     

Distribution and habitats of <Emphasis Type="Italic">Phengaris</Emphasis> (<Emphasis Type="Italic">Maculinea</Emphasis>) butterflies and population ecology of <Emphasis Type="Italic">Phengaris</Emphasis><Emphasis Type="Italic">teleius</Emphasis> in China

Many species of the butterfly genus Phengaris are regarded as endangered in many parts of their distribution. Several species are also widely distributed across northern China. Due to land use change and overgrazing, their habitats are declining and many patches have been lost. This paper investigates the distribution and habitats of the Chinese Phengaris species (of the subgenus Maculinea). Shrub-grassland near forests seem the most frequent habitat for Phengaris, while flat open grasslands are mostly over-grazed and thus survival for Phengaris butterflies there seems difficult. Throughout Europe, P. teleius is an endangered species, while there is still no information on its status in China. To improve the knowledge on the population ecology of P. teleius, its population structure, adult behaviour and movement were studied through mark–release–recapture methods in the Qinling Mountains of Taibai County. Eight grassland patches which were potentially suitable were found in the area in 2013. In total, 480 individuals (274 females) were marked, resulting in an overall recapture rate of 16 %. The average daily population size was 44 butterflies (±23 SD) during the adult flight period. Sixty-seven percent of the females and 38 % of the males moved less than 50 m, and 17 % of recaptured females and 38 % of males moved more than 200 m. The mean movement distance was 107 ± 177 m for males and 182 ± 122 m for females. The majority of the recaptures (86 %) were made within the patches, only a few individuals (14 %) moved between patches. Due to human disturbance and destruction, all of the eight potentially suitable patches are becoming smaller and increasingly isolated, thus these populations of P. teleius may face an increasing risk of extinction, which may well be a tip of the iceberg of habitat loss and fragmentation of P. teleius in Taibai County and possibly beyond. Hence we hope our initial study of P. teleius could have positive impacts on the conservation of Phengaris butterflies in China.     

Growth and phosphorus removal by <Emphasis Type="Italic">Synechococcus elongatus</Emphasis> co-immobilized in alginate beads with <Emphasis Type="Italic">Azospirillum brasilense</Emphasis>

This study examined the co-immobilization of the cyanobacterium Synechococcus elongatus with the plant growth-promoting bacterium Azospirillum brasilense in alginate beads and its potential application for the removal of phosphorus from aquaculture wastewater. Co-immobilization of both microorganisms significantly increased the cell density of S. elongatus (2852.5?×?10⁴ cells mL^?1) compared with that of immobilization of cyanobacteria alone (1325.2?×?10⁴ cells mL^?1). Chlorophyll a content was similar in co-immobilized (11.1?±?3.5 pg cell^?1) and immobilized S. elongatus (14.5?±?4.9 pg cell^?1). Azospirillum brasilense showed continuous growth until day 2, after which its cell concentration declined until the end of the assay. Co-immobilized S. elongatus removed more phosphorus (44.8 %) than immobilized cyanobacteria cells alone (32.0 %). In conclusion, phosphate removal was greater with free cells of S. elongatus but overlapped with the values that were obtained with the treatment of co-immobilization of cells. Our results demonstrate that A. brasilense enhances the growth of S. elongatus and improves its removal of phosphorus when they are co-immobilized in alginate beads compared with only immobilization of cyanobacteria cells alone.     

A homomeric geranyl diphosphate synthase-encoding gene from <Emphasis Type="Italic">Camptotheca acuminata</Emphasis> and its combinatorial optimization for production of geraniol in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Geranyl diphosphate (GPP), the unique precursor for all monoterpenoids, is biosynthesized from isopentenyl diphosphate and dimethylallyl diphosphate via the head-to-tail condensation reaction catalyzed by GPP synthase (GPPS). Herein a homomeric GPPS from Camptotheca acuminata, a camptothecin-producing plant, was obtained from 5′- and 3′-rapid amplification of cDNA ends and subsequent overlap extension and convenient PCR amplifications. The truncate CaGPPS was introduced to replace ispA of pBbA5c-MevT(CO)-MBIS(CO, ispA), a de novo biosynthetic construct for farnesyl diphosphate generation, and overexpressed in Escherichia coli, together with the truncate geraniol synthase-encoding gene from C. acuminata (tCaGES), to confirm CaGPPS-catalyzed reaction in vivo. A 24.0 ± 1.3 mg L^?1 of geraniol was produced in the recombinant E. coli. The production of GPP was also validated by the direct UPLC-HRMS^E analyses. The tCaGPPS and tCaGES genes with different copy numbers were introduced into E. coli to balance their catalytic potential for high-yield geraniol production. A 1.6-fold increase of geraniol production was obtained when four copies of tCaGPPS and one copy of tCaGES were introduced into E. coli. The following fermentation conditions optimization, including removal of organic layers and addition of new n-decane, led to a 74.6 ± 6.5 mg L^?1 of geraniol production. The present study suggested that the gene copy number optimization, i.e., the ratio of tCaGPPS and tCaGES, plays an important role in geraniol production in the recombinant E. coli. The removal and addition of organic solvent are very useful for sustainable high-yield production of geraniol in the recombinant E. coli in view of that the solubility of geraniol is limited in the fermentation broth and/or n-decane.     

Development and characterization of <Emphasis Type="Italic">japonica</Emphasis> rice lines carrying the brown planthopper-resistance genes <Emphasis Type="Italic">BPH12</Emphasis> and <Emphasis Type="Italic">BPH6</Emphasis>

The brown planthopper (Nilaparvata lugens Stål; BPH) has become a severe constraint on rice production. Identification and pyramiding BPH-resistance genes is an economical and effective solution to increase the resistance level of rice varieties. All the BPH-resistance genes identified to date have been from indica rice or wild species. The BPH12 gene in the indica rice accession B14 is derived from the wild species Oryza latifolia. Using an F₂ population from a cross between the indica cultivar 93-11 and B14, we mapped the BPH12 gene to a 1.9-cM region on chromosome 4, flanked by the markers RM16459 and RM1305. In this population, BPH12 appeared to be partially dominant and explained 73.8% of the phenotypic variance in BPH resistance. A near-isogenic line (NIL) containing the BPH12 locus in the background of the susceptible japonica variety Nipponbare was developed and crossed with a NIL carrying BPH6 to generate a pyramid line (PYL) with both genes. BPH insects showed significant differences in non-preference in comparisons between the lines harboring resistance genes (NILs and PYL) and Nipponbare. BPH growth and development were inhibited and survival rates were lower on the NIL-BPH12 and NIL-BPH6 plants compared to the recurrent parent Nipponbare. PYL-BPH6 + BPH12 exhibited 46.4, 26.8 and 72.1% reductions in population growth rates (PGR) compared to NIL-BPH12, NIL-BPH6 and Nipponbare, respectively. Furthermore, insect survival rates were the lowest on the PYL-BPH6 + BPH12 plants. These results demonstrated that pyramiding different BPH-resistance genes resulted in stronger antixenotic and antibiotic effects on the BPH insects. This gene pyramiding strategy should be of great benefit for the breeding of BPH-resistant japonica rice varieties.     

Complete chloroplast genome sequence of <Emphasis Type="Italic">Capsicum</Emphasis><Emphasis Type="Italic">baccatum</Emphasis> var. <Emphasis Type="Italic">baccatum</Emphasis>

Molecular markers derived from the complete chloroplast genome can provide effective tools for species identification and phylogenetic resolution. Complete chloroplast (cp) genome sequences of Capsicum species have been reported. We herein report the complete chloroplast genome sequence of Capsicum baccatum var. baccatum, a wild Capsicum species. The total length of the chloroplast genome is 157,145 bp with 37.7 % overall GC content. One pair of inverted repeats, 25,910 bp in length, was separated by a small single-copy region (17,974 bp) and large single-copy region (87,351 bp). This region contains 86 protein-coding genes, 30 tRNA genes, 4 rRNA genes, and 11 genes contain one or two introns. Pair-wise alignments of chloroplast genome were performed for genome-wide comparison. Analysis revealed a total of 134 simple sequence repeat (SSR) motifs and 282 insertions or deletions variants in the C. baccatum var. baccatum cp genome. The types and abundances of repeat units in Capsicum species were relatively conserved, and these loci could be used in future studies to investigate and conserve the genetic diversity of the Capsicum species.     

Enhancing carotenoid production in <Emphasis Type="Italic">Rhodotorula mucilaginosa</Emphasis> KC8 by combining mutation and metabolic engineering

Rhodotorula mucilaginosa has been considered as a potential industrial yeast due to its unicellular and fast-growing characteristics, and its ability to produce carotenoids, including torularhodin. However, its low total carotenoid production limits its commercial application. In this study, mutation breeding and metabolic engineering were employed to enhance carotenoid production in the R. mucilaginosa strain KC8. After chemical–physical mutagenesis, R. mucilaginosa K4 with a 67% greater concentration of carotenoids (14.47 ± 0.06 mg L^?1) than R. mucilaginosa KC8 (8.67 ± 0.07 mg L^?1) was obtained. To further enhance carotenoid production, gene HMG1 encoding the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase was introduced from another yeast, Saccharomyces cerevisiae, and overexpressed in R. mucilaginosa K4. The carotenoid production of HMG1-gene-overexpression transformant G1 reached 16.98 mg L^?1. To relieve the feedback inhibition of ergosterol, and to down-regulate ergosterol synthesis, ketoconazole, an ergosterol synthesis inhibitor, was added at a concentration of 28 mg L^?1. The carotenoid production of the transformant G1 reached 19.14 ± 0.09 mg L^?1, which was 121% higher than in R. mucilaginosa KC8. This suggests that a combination of chemical–physical mutagenesis, overexpression of the HMG1 gene, and adding ketoconazole is an effective strategy to improve carotenoid production.     

Molecular cytogenetic characterization of <Emphasis Type="Italic">Triticum timopheevii</Emphasis> chromosomes provides new insight on genome evolution of <Emphasis Type="Italic">T. zhukovskyi</Emphasis>

Triticum timopheevii (2n = 4x = 28, GGA^tA^t) is a tetraploid wheat formerly cultivated in western Georgia. The natural allopolyploid Triticum zhukovskyi is a hexaploid taxon originated from hybridization of T. timopheevii with cultivated einkorn T. monococcum (2n = 2x = 14, A^mA^m). Karyotypically T. timopheevii and T. zhukovskyi differ from other tetraploid and hexaploid wheats and were assigned to the section Timopheevii of the genus Triticum L. Triticum timopheevii and T. zhukovskyi are resistant to many fungal diseases and therefore could potentially be utilized for wheat improvement. We were aiming to precisely identify all T. timopheevii chromosomes and to trace the evolution of T. zhukovskyi. For this, we developed a set of molecular cytogenetic landmarks based on eleven DNA probes. Each chromosome can now be characterized by two to eight probes. The pTa-535 sequence allows the identification of all A^t-genome chromosomes, whereas G-genome and some A^t-genome chromosomes can be identified using (GAA/CTT) _n and pSc119.2 probes. The probes pAesp_SAT86, pAs1, Spelt-1, Spelt-52 and 5S and 45S rDNA can be applied as additional markers to discriminate particular chromosomes or chromosomal regions. The distribution of (GAA/CTT) _n , pTa-535 and pSc119.2 DNA probes on T. timopheevii chromosomes is distinct from other tetraploid wheats and can therefore be used to track individual chromosomes in introgression programs. Our study confirms the origin of T. zhukovskyi from hybridization of T. timopheevii with T. monococcum; however, we show that the emergence was accompanied by changes involving mostly A^t-genome chromosomes. This may be due to the presence of two closely related A-genomes in the T. zhukovskyi karyotype.     

Cytogenetic characterization of <Emphasis Type="Italic">Amaranthus caudatus</Emphasis> L. and <Emphasis Type="Italic">Amaranthus hybridus</Emphasis> subsp<Emphasis Type="Italic">. cruentus</Emphasis> (L.) Thell.

The present study is aimed to identify genetic variability between two species of Amaranthus viz., A. caudatus and A. hybridus subsp. cruentus, two economically important species, cultivated mainly for grain production. Karyomorphological studies in Amaranthus are scarce, probably due to higher number of small sized chromosomes. Karyomorphological studies were conducted using mitotic squash preparation of young healthy root tips. Karyological parameters and karyotypic formula were established using various software programs and tabulated the karyomorphometric and asymmetry indices viz., Disparity index, Variation coefficient, Total forma percentage, Karyotype asymmetry index, Syi index, Rec index, Interchromosomal and Intrachromosomal asymmetry index and Degree of asymmetry of karyotypes. The mitotic chromosome number observed for A. caudatus was 2n = 32 with a gametic number n = 16 and A. hybridus subsp. cruentus was 2n = 34 with a gametic number n = 17. In A. caudatus the chromosome length during somatic metaphase ranged from 0.8698 to 1.7722 μm with a total length of 39.1412 μm. In A. hybridus subsp. cruentus the length of chromosome ranged from 0.7756 to 1.9421 μm with a total length of 44.9922 μm. Various karyomorphometry and asymmetry indices analyzed revealed the extend of interspecific variation and their evolutionary status.     

Interactions of Algae within the Community of <Emphasis Type="Italic">Gracilaria gracilis</Emphasis> (Rhodophyta)

Interactions among the unattached red alga Gracilaria gracilis, the dominant species of an algal community, and associated algal species Chaetomorpha linum, Enteromorpha prolifera f. prolifera, and Polysiphonia sp. were studied during and after an algal bloom. It was shown that during their bloom the associated algae Enteromorpha and Polysiphonia sp. significantly decreased the photosynthetic rate of G. gracilis but did not affect its growth rate. It is suggested that the inhibition of Gracilaria gracilis photosynthesis is connected to the impact of extracellular metabolites excreted by Chaetomorpha linum, Enteromorpha prolifera f. prolifera, and Polysiphonia sp. In laboratory experiments, the photosynthetic rate of the associated species was enhanced in the presence of Gracilaria. However, no significant alterations were observed in the content of chlorophyll a, growth, and the dark respiration rates of associated algae when they were kept together with Gracilaria. It was suggested that allelopathic interactions observed among algal species during the formation of the monospecific Gracilaria community, as well as during algal blooms, are not determinative.