Quantification and characterization of nuclear genomes in commercial red seaweeds: Gracilariales and Gelidiales

Nuclear genome profiles were developed for representative species of the Gelidiales and Gracilariales using information from present and previous studies of cytogenetics, cytophotometry and DNA reassociation kinetics. Results indicate that species of Gracilaria and Gracilariopsis are characterized by distinct chromosome complements of n = 24 and n = 32, respectively, a narrow range of small genome sizes (2C = 0.35–0.45 pg) and a wide range of values for repeated and unique genome sequences. In contrast, the Gelidiales investigated are characterized by a wide range of chromosome complements, n = 6–29, a wider range of genome sizes (2C = 0.42–0.68 pg) and a large proportion of unique genome sequences. Nuclear genome sizes for species of the Gelidiales and Gracilariales are compared with estimates of other red algal orders including the Bangiales, Ceramiales and Gigartinales.     

Agar Analysis,nuclear genome quantification and characterization of four agarophytes (Gracilaria) from the Mexican Gulf Coast

DNA reassociation kinetics were used to determine nuclear genome organization and complexity in four species of Gracilaria (Gracilariales, Rhodophyta). In Gracilaria tikvahiae, G. caudata, G. cervicornis and G. divaricata, results indicate the presence of three second order components corresponding to fast, intermediate and slow fractions. Repetitive sequences varied from 13–46% and unique DNA ranged from 45–78%, Thermal denaturation (T _m) indicated guanine + cytosine (G + C) levels of 41.9–46.0 mol % G + C. Microspectrophotometry with the DNA-localizing fluorochrome DAPI was used to quantify nuclear DNA content. Comparisons of mean nuclear DNA (I _f) values to chicken erythrocytes (RBC) resulted in an estimate of 0.37–0.40 pg/2C genomes for the four Gracilaria species. Total agar content following alkaline pretreatment ranged from 7–15% dry weight. Gel strengths were generally below commercial levels, ranging from 40–260 g cm⁻² Nuclear genome profiles developed from information for genome size, organization and complexity are compared with data for agar quantity and quality. Gel quality and quantity do not appear to be correlated with either large repetitive fraction DNA or a high degree of genome complexity as previously speculated.     

DNA base composition heterogeneity in some agarophytes (Gracilariales,Rhodophyta) from Mexico and the Philippines

Estimates of nuclear DNA base composition by determination of thermal denaturation temperatures (T_m) indicate guanine + cytosine (G + C) levels of 35.4–46.8% for ten species of the Gracilariaceae, representing the generaGracilaria andHydropuntia. T_m values were found to be reproducible with variation among most samples and replicates of less than 1 °C and 2 mol%. Interspecific variation in G + C values was less than 11.4% amongGracilaria species. Calculation of intragenomic base pair composition distribution based on mid-resolution thermal denaturation (A 1 °C/min with 4s interval H and dT logging) indicated an inverse relationship between maximum similarity values and taxonomic rank. Intraspecific (population level) maximum similarity (homology) values were estimated to range from 79–90% inGracilaria tikvahiae (4 isolates). Interspecific values of 46–69% were found in 13 species ofGracilaria. Nucleotide distribution similarity values for the Gracilariaceae are compared with previous information for genome organization and complexity, genome size and karyotype patterns.Author for correspondence     

Karyology,nuclear genome quantification and characterization of the carrageenophytesEucheuma andKappaphycus (Gigartinales)

DNA reassociation kinetics were used to determine nuclear genome organization and complexity in the carrageenophyteKappaphycus alvarezii. Results indicate the presence of three second order components corresponding to fast (12%), intermediate (38%) and slow (50%) fractions. Microspectrophotometry with the DNA-localizing fluorochrome DAPI confirmed ploidy level differences in the gametophytic and tetrasporophytic phases. Comparison of mean nuclear DNA (I _f ) values to chicken erythrocytes (RBC) resulted in pg/2C genome estimates:Eucheuma denticulatum=0.35,E. isiforme=0.44,Kappaphycus alvarezii=0.32 andK. striatum=0.42. Karyological studies of tetraspore mother cells during diakinesis using aceto-orcein revealed a chromosome complement of 10 forEucheuma denticulatum andKappaphycus alvarezii.     

Agar quality of commercial agarophytes from different geographical origins: 1. Physical and rheological properties

Six economically important species ofGracilaria, from a number of commercial sources around the world, andGracilariopsis lemaneiformis, collected from two Japanese localities, were used as the sources of raw material for the evaluation of agar quality. Agar-agar was extracted by pretreatment with various concentratrions of NaOH (0%, 3%, 5%, 7%, 10%) incubated at 80 °C for 2 h. Agar yield, viscosity, dynamic gelling and melting temperature and gel texture were determined for 1.5% agar gels. The highest agar yield was obtained fromG. gracilis from Argentina (39.5%), while the lowest was from BrazilianG. gracilis (13.37%). Dynamic gelling temperature was highest in the agar fromG. gracilis from Turkey (59 °C) and lowest in the non-alkali treated agar isolated fromG. edulis from Indonesia (46 °C). Melting temperature ranged from 96 °C in the agars from the JapaneseGracilariopsis andG. chilensis from Chile to 69 °C in the non-alkali treated agar fromG. edulis from Indonesia. In general, all species produced an agar with high gel strength after treatment with 5% NaOH, except forG. chilensis and the twoGracilariopsis species, which produced an agar with high gel strength after treatment with 3, 7 and 10% NaOH. The highest gel strength (2056 ± 13.6 cm^–2) and hardest gel (261 ± 19.89 g mm^–2) were obtained fromG. lemaneiformis from Japan (Oita Prefecture) after treatment with 7 and 10% NaOH respectively. The lowest gel strength (351 ± 93 cm^–2) was obtained fromG. gracilis from Brazil after treatment with 3% NaOH. The softest gel (66.31 ± 9.63 g mm^–2) was isolated fromG. tenuistipitata from China, after treatment with 3% NaOH. The most flexible gel (11.62 ± 0.31 g mm^–2 × 10²) was obtained fromG. chilensis from Chile after treatment with 3% NaOH.Author for correspondence     

Nuclear genome characterization and carrageenan analysis ofGymnogongrus griffithsiae (Rhodophyta) from North Carolina

DNA reassociation kinetics were used to determine nuclear genome organization and complexity inGymnogongrus griffithsiae (Gigartinales, Rhodophyta). Results indicate the presence of three second order components corresponding to fast (3%), intermediate (8%) and slow (89%) fractions. Thus the genome consists mainly of unique sequences. Thermal denaturation (T _m) indicated a nuclear DNA base pair composition of 40 mol% G + C. Microspectrophotometry with the DNA-localizing fluorochrome DAPI was used to confirm ploidy level differences in the gametophytic and tetrasporoblastic phases. Comparisons of mean nuclear DNA (I _f) values to chicken erythrocytes (RBC) resulted in an estimate of 0.32 pg/2 C genome forGymnogongrus griffithsiae. Karyological studies using aceto-orcein revealed the presence of ca. 23 bivalents during diakinesis of tetrasporangial mother cells. Total carrageenan content in water extraction was 30% dry weight. Infrared spectroscopy confirmed the isolated carrageenan to be the iota-fraction.     

Nuclear genome characterization of the carrageenophyteAgardhiella subulata (Rhodophyta)

DNA reassociation kinetics were used to determine nuclear genome organization and complexity inAgardhiella subulata (Gigartinales, Rhodophyta). Results indicate the presence of three second-order components corresponding to fast (22%), intermediate (68%) and slow (10%) fractions. Thus, the genome consists of 90% repetitive sequences. Microspectrophotoometry with the DNA-localizing fluorochrome DAPI was used to confirm ploidy level differences in the gametophytic and tetrasporophytic phases. Results indicate that meiosis occurs during tetrasporogenesis. Comparison of mean nuclear DNA (I_f) values to chicken erythrocytes (RBC) resulted in an estimate of 0.9 pg/2C genome forAgardhiella. Karyological studies using aceto-orcein revealed a chromosome complement of 2N = 44 in carposporangia and the presence of 22 bivalents during diakinesis of tetraspore mother cells.     

Nuclear genome characterization of the carrageenophyteHypnea musciformis (Rhodophyta)

DNA reassociation kinetics were used to determine nuclear genome organization and complexity in the carrageenophyteHypnea musciformis (Gigartinales, Rhodophyta). Results indicate the presence of three second order components corresponding to fast (2%), intermediate (33%) and slow (65%) fractions. Microspectrophotometry with the DNA-localizing fluorochrome DAPI confirmed ploidy level differences in the gametophytic and tetrasporophytic phases. Elevated (endopolyploid) nuclear DNA levels were observed in both gametophytic and cystocarpic tissue. Comparison of mean nuclear DNA (If) values to chicken erthrocytes (RBC) resulted in an estimate of 0.22 pg/2 C genome forHypnea musciformis. Karyological studies using aceto-orcein revealed a chromosome complement of five bivalents during diakinesis of tetraspore mother cells.     

Unraveling the nuclear and chloroplast genomes of an agar producing red macroalga, Gracilaria changii (Rhodophyta,Gracilariales)

Agar and agarose have wide applications in food and pharmaceutical industries. Knowledge on the genome of red seaweeds that produce them is still lacking. To fill the gap in genome analyses of these red algae, we have sequenced the nuclear and organellar genomes of an agarophyte, Gracilaria changii. The partial nuclear genome sequence of G. changii has a total length of 35.8 Mb with 10,912 predicted protein coding sequences. Only 39.4% predicted proteins were found to have significant matches to protein sequences in SwissProt. The chloroplast genome of G. changii is 183,855 bp with a total of 201 open reading frames (ORFs), 29 tRNAs and 3 rRNAs predicted. Five genes: ssrA, leuC and leuD CP76_p173 (orf139) and pbsA were absent in the chloroplast genome of G. changii. The genome information is valuable in accelerating functional studies of individual genes and resolving evolutionary relationship of red seaweeds.     

Characterization of an agar fraction extracted from Gracilaria dura (Gracilariales,Rhodophyta)

The evaluation of biopolymers of biotechnological interest has stressed the importance of renewable sources from the northern Adriatic Sea. In the frame of this research program, the red alga Gracilaria cf. dura, has been studied. Agar was obtained from Gracilaria cf. dura by means of alkali treatment and hot water extraction. In order to purify the extracts, amylase and isopropyl alcohol were used. Analysis of sugars was carried out by means of gas chromatography on alditol acetate derivatives; sulfate content was evaluated by means of infrared spectroscopy. An analysis of molecular weight distribution was carried out by means of high performance gel permeation chromatography coupled with a low angle laser light scattering device as detector. Analytical data were examined in terms of specificity of the algal source utilized, and the agar fractions extracted by means of this procedure were compared with a commercial sample.     

Cultivation of Gracilaria verrucosa (Gracilariales,Rhodophyta) Strain G-16 for agar

The agarophyte, Gracilaria verrucosa Strain G-16, has been grown in sustained cultivation over a period of five years. During this period, a number of experiments were conducted to examine the productivity, agar yield and agar gel strength of this strain. Productivities range from 3–31 g dry wt m^–2 d^–1 and are generally highest in the summer when annual water temperatures and daily irradiances are highest. In the summer months agar yield from Strain G-16 appears to be lowest whereas the gel strength of the agars was highest (> 750 g cm^–2).     

The yield,physical and chemical properties of agar gel from Gracilaria species (Gracilariales,Rhodophyta) of the Kenya coast

Six species of Gracilaria, G. corticata J. Agardh, G. crassa Harvey, G. millardetii J. Agardh, G. salicornia (J. Ag.) Dawson, G. verrucosa (Huds.) Papenfuss and Gracilaria sp, collected from different stations along the Kenya coast were studied. The yield of hot water native agar extract ranges from 8.1–30% of dry weight, with G. verrucosa and G. salicornia having the greatest and the least yield, respectively. The gel-strength of 1.5% agar solution was highest in G. verrucosa (220 g cm^–2) and lowest in G. corticata (< 60 g cm – 2) whereas the highest gelling temperature was recorded for Gracilaria sp. (40.4 °C) and the lowest in G. verrucosa (28.9 °C). 3,6 anhydrogalactose content was the highest in G. verrucosa and the lowest in G. corticata while sulphate content was higher in the latter.     

Studies on the yield and gel strength of agar from Gracilaria domingensis Sonder ex Kuetzing (Gracilariales,Rhodophyta) following the addition of calcium

Studies were carried out on the seasonal variation in yield and gel strength of agar from Gacilaria domingensis with and without the addition of calcium chloride. Extraction was done with and without treatment with 1% hydrochloric acid. The results showed an increase in yield and gel strength when an alkaline solution of calcium was used, but the gel strength was low. For commercial use, Gracilaria domingensis should be mixed with better quality Gracilaria species because of its low gel strength.     

Ultrastructure of the cell wall of Gracilaria cf. verrucosa (Gracilariales,Rhodophyta): effects of steam explosion

Ultrastructural (SEM, TEM) and cytochemical organization of the skeletal and matrix polysaccharides of fresh and steam exploded Gracilaria verrucosa thalli were compared to chemical analyses of the agar extracted after this treatment. Upon steam explosion, surface microfractures appeared in limited areas of the cortex. SEM preparations indicated that apical and medullary regions were the most affected, resulting in detachment of adjacent cells. In TEM sections, the cuticle of the surface layer exhibited deeply altered zones with a spongy appearance. In the cytoplasm starch grains were disrupted. The skeletal component of the immediate cell wall was maintained with a different organization of the microfibrils and was positive to periodic acid-thiosemicarbazide-silver proteinate. The intercellular matrix contained granules and fibrils and lacked a ruthenium red reaction. These results are consistent with lower sulfate-ester and higher glucose contents detected in agars extracted using steam explosion.     

Seasonality of some species ofGracilaria (Gracilariales,Rhodophyta) from Kenya

Studies on the seasonality of 5Gracilaria species (G. corticata, G. crassa, G. millardetii, G. salicornia, G. verrucosa) were carried out. The seasonal abundance of the various species varied with time and geographical location. Nevertheless, a general single peak was evident between the months of September and December.G. verrucosa on the other hand had its peak biomass in July/August.G. millardetti is the only species which apparently preferred lower salinities; the rest of the species had no special trends as far as salinity is concerned. The occurrence ofG. verrucosa for a very short period during the cool part of the year is attributed to the high temperature that prevails in the coastal waters.     

Content of arachidonic and eicosapentaenoic acids in polar lipids from Gracilaria (Gracilariales,Rhodophyta)

Fatty acid composition, especially the distribution of eicosapolyenoic acids in several species of Gracilaria, was analyzed in relation to their taxonomy. The species have been grouped into two types based on distribution of these polyenoic acids: Type 1, which contains palmitic, oleic and arachidonic acids as the major components, and Type II, which contains eicosapentaenoic acid in addition to Type I fatty acids. Octadecapolyenoic acids were detected only in trace amounts in each Type. A similar remarkable difference also was observed in the fatty acid composition of lipid classes. The major component of eicosapolyenoic acids in Type I was arachidonic acid in all lipid classes. In Type II, eicosapentaenoic acid was the major component in monogalactosyl diacylglycerol, digalactosyl diacylglycerol, sulfoquinovosyl diacylglycerol and phosphatidylglycerol. Arachidonic and eicosapentaenoic acids were contained in large amounts in Type II phosphatidylcholine. Grouping of Gracilaria species into Type I and Type II is not entirely consistent with morphological and taxonomic features, but the difference in fatty acid composition is likely due to genetic rather than to environmental factors.     

Organellar DNA restriction fragment length polymorphism (RFLP) and nuclear random amplified polymorphic DNA (RAPD) analyses of morphotypes of Gracilaria (Gracilariales,Rhodophyta) from Chile

The extreme phenotypic variability recognized among the species of Gracilaria has highlighted the need for the application of refined methods to help solve taxa identifications. In Chile, there still exists uncertainty about the exact number of Gracilaria species. Our investigations are centered on DNA analyses of morphotypes collected from different geographical locations, namely Lenga and Isla Santa María, Region VIII (36°00 S to 38°00 S), and Maullín, Region X (39°30 S to 43°40 S). These two regions of Chile are considered as areas of confluence of G. chilensis, G. verrucosa, and a species of Gracilariopsis. In this study four morphotypes, from a natural bed located in Maullín, were analyzed for RFLP of plastid DNA and the results compared with data of four morphotypes from a bed in Lenga. The DNA banding patterns from each enzyme digest were identical irrespective of morphotypes and/or locations. In an attempt to unravel the nature of the morphological differences found among Lenga and Maullín morphotypes, RAPD analyses of nuclear DNA were also performed; however, no polymorphism has been found yet. Therefore, the data of this study, as well as concurrent data from preliminary interfertility tests, suggest that all morphotypes belong to a single taxon, Gracilaria chilensis.Departamento de Botánica, Facultad de Ciencias Naturales y Oceanográficas     

Differentiation of Male Gametes in Gracilaria verrucosa (Rhodophyta, Gracilariales)

The development of male gametes (spermacia) in the red alga Gracilaria verrucosa has been studied using methods of transmission electron microscopy. Early spermatangia located along the wall of the conceptacle show an elongated shape in the thin sections. In the central part of the electron-dense cytoplasm of these cells there is a nucleus; numerous fibrous vesicles are arranged in the periphery. During the process of differentiation, the spermatangia become more rounded in shape and a large spermatangial vesicle is developed. The subsequent development of spermatium is accompanied by polarization of the spermatangium and the subsequent excretion of the spermatangial vesicle. The spermatia are oval cells containing a nucleus and fibrous vesicles. The process of differentiation of male gametes in G. verrucosa does not differ from that in five species of the genus Gracilaria, where it has already been studied. However, any conclusions about the degree of similarity between the spermatia in all the studied species can be made only after a detailed comparative analysis of the ultrastructural characteristics of these gametes.     

A seasonal study of the red seaweeds Solieria tenera and three species of Gracilaria from Jamaica

Three species of Gracilaria viz G. cervicornis, G. domingensis, G. verrucosa and Solieria tenera (Gigartinales, Rhodophyta) have been studied for their monthly variation in dry wt yields, ash, soluble carbohydrate, protein, lipid and insoluble carbohydrate contents for one year. The dry wt yields were higher in G. domingensis. Soluble carbohydrate and ash contents showed an inverse relationship in all the species. Protein content was lower, below 5% of the dry wt for all the species. A comparison of protein:carbohydrate ratios showed a similarity between morphologically similar G. domingensis and G. cervicornis in that there was less variation in the ratio. From this, it is assumed that flattened morphology probably is more efficient in maintaining nutrient balance to keep up the growth rates.