Identification of 27 Porphyra lines (Rhodophyta) by DNA fingerprinting and molecular markers

Twenty-seven Porphyra lines, including lines widely used in China, wild lines and lines introduced to China from abroad in recent years, were screened by random amplified polymorphic DNA (RAPD) technique with 120 operon primers. From the generated RAPD products, 11 bands that showed stable and repeatable RAPD patterns amplified by OPC-04, OPJ-18 and OPX-06, respectively were scored and used to develop the DNA fingerprints of the 27 Porphyra lines. Moreover, the DNA fingerprinting patterns were converted into computer language expressed with two digitals, 1 and 0, which represented the presence (numbered as 1) or absence (numbered as 0) of the corresponding band, respectively. Based on the above results, computerized DNA fingerprints were constructed in which each of the 27 Porphyra lines has its unique fingerprinting pattern and can be easily distinguished from others. Software named PGI (Porphyra germplasm identification) was designed for identification of the 27 Porphyra lines. In addition, seven specific RAPD markers from seven Porphyra lines were identified and two of them were successfully converted into SCAR (sequence characterized amplification region) markers. The developed DNA fingerprinting and specific molecular markers provide useful ways for the identification, classification and resource protection of the Porphyra lines.

Construction of AFLP-based genetic linkage map for Zhikong scallop, Chlamys farreri Jones et Preston and mapping of sex-linked markers

Zhikong scallop (Chlamys farreri) is an economically important aquaculture species in China; however, frequent mass mortality seriously affects the development of its industry. Genetic linkage map is useful for genetic improvement and selective breeding of C. farreri. Linkage maps were constructed using an intraspecific F-1 cross and amplified fragment length polymorphism (AFLP) markers. Thirty-two selected AFLP primer combinations produced 545 AFLP markers that were polymorphic in either of the parents and segregated in the progeny. Of these segregating markers, 166 were mapped to 19 linkage groups of the female framework map, covering a total of 1503.9 cM, with an average marker spacing of 10.2 cM; and 197 markers were assigned to 20 linkage groups of the male map, covering a total of 1630.7 cM, with 9.2 cM per marker. A sex-linked marker was mapped on the female map with zero recombination and a LOD of 27.3. The genetic length of C farreri genome was estimated as 1889.0 cM for the female and 1995.9 cM for the male. The coverage of the framework map was calculated as 79.6% for the female and 81.7% for the male. When the triplets and doublets were considered, the observed length of the map was calculated as 1610.2 cM with coverage of 85.2% for the female, and 1880.5 cM with coverage of 94.2% for the male. The genetic maps presented here will serve as a basis for the construction of a high-resolution genetic map and mapping of economically important genes. (C) 2004 Published by Elsevier B.V.

DNA fingerprinting of selected Laminaria (Phaeophyta) gametophytes by RAPD markers

Random amplified polymorphism DNA (RAPD) analysis was applied to germplasm characterization in 33 different selected Laminaria male and female gametophytes. The positional homology of the RAPD analysis using sequence characterized applied region (SCAR) method was successfully conducted. A total of 233 polymorphic loci were obtained from 18 selected primers after screening, of which 27 stable and clear bands were selected to construct a fingerprint map for discrimination of each gametophyte. Seven RAPD markers from five primers were finally determined by a computer program to construct the fingerprint map. Three specific markers closely related with gametophytes were obtained and were converted to gametophytic SCAR markers, the first SCAR marker report on Laminaria germplasm and applicable to cultivars identification. These results demonstrated the feasibility of applying RAPD markers to germplasm characterization in selected Laminaria gametophytes, and can provide a molecular basis for breeding new Laminaria strains. (C) 2004 Elsevier B.V. All rights reserved.

Molecular identification of 16 Porphyra lines using sequence-related amplified polymorphism markers

Sequence-related amplified polymorphism (SRAP) is a novel molecular marker technique designed to amplify open reading frames (ORFs). The SRAP analytic system was set up and applied to Porphyra germplasm identification in this study for the first time. Sixteen Porphyra lines were screened by SRAP technique with 30 primer combinations. In the analysis, 14 primer combinations produced stable and reproducible amplification patterns in three repetitive experiments. Among the total 533 amplified fragments, 522 (98%) were polymorphic, with an average of 38 fragments for each primer combination, ranging in size from 50 to 500 bp. The 533 fragments were visually scored one by one and then used to develop a dendrogram with Unweighted Pair-Group Method Arithmetic Average (UPGMA), and the 16 Porphyra lines were divided into two major groups at the 0.68 similarity level. From the total 533 fragments, I I amplified by two primer combinations, ME1/EM1 and ME4/EM6, were used to develop the DNA fingerprints of the 16 Porphyra lines. The DNA fingerprints were then converted into binary codes, with I and 0 representing presence and absence of the corresponding amplified fragment, respectively. In the DNA fingerprints, each of the 16 Porphyra lines has its unique binary code and can be easily distinguished from the others. This is the first report on the development of SRAP technique and its utilization in germplasm identification of seaweeds. The results demonstrated that SRAP is a simple, stable, polymorphic and reproducible molecular marker technique for the classification and identification of Porphyra lines. (c) 2007 Elsevier B.V. All rights reserved.

A genetic linkage map of Pacific white shrimp (Litopenaeus vannamei): sex-linked microsatellite markers and high recombination rates

Pacific white shrimp (Litopenaeus vannamei) is the leading species farmed in the Western Hemisphere and an economically important aquaculture species in China. In this project, a genetic linkage map was constructed using amplified fragment length polymorphism (AFLP) and microsatellite markers. One hundred and eight select AFLP primer combinations and 30 polymorphic microsatellite markers produced 2071 markers that were polymorphic in either of the parents and segregated in the progeny. Of these segregating markers, 319 were mapped to 45 linkage groups of the female framework map, covering a total of 4134.4 cM; and 267 markers were assigned to 45 linkage groups of the male map, covering a total of 3220.9 cM. High recombination rates were found in both parental maps. A sex-linked microsatellite marker was mapped on the female map with 6.6 cM to sex and a LOD of 17.8, two other microsatellite markers were also linked with both 8.6 cM to sex and LOD score of 14.3 and 16.4. The genetic maps presented here will serve as a basis for the construction of a high-resolution genetic map, quantitative trait loci (QTLs) detection, marker-assisted selection (MAS) and comparative genome mapping.

Genetic structure analysis of natural Sargassum muticum (Fucales, Phaeophyta) populations using RAPD and ISSR markers

Sargassum muticum is important in maintaining the structure and function of littoral ecosystems, and is used in aquaculture and alginate production, however, little is known about its population genetic attributes. In this study, random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were used to investigate the genetic structure of four populations of S. muticum and one outgroup of S. fusiforme (Harv.) Setchell from Shandong peninsula of China. The selected 24 RAPD primers and 19 ISSR primers amplified 164 loci and 122 loci, respectively. Estimates of genetic diversity with different indicators (P%, percentage of polymorphic loci; H, the expected heterozygosity; I, Shannon's information index) revealed low or moderate level of genetic variations within each S. muticum population, and a high level of genetic differentiations were determined with pairwise unbiased genetic distance (D) and fixation index (F-ST ) among the populations. The Mantel test showed that two types of matrices of D and F-ST were highly correlated whether from RAPD (r = 0.9706, P = 0.009) or ISSR data (r = 0.9161, P = 0.009). Analysis of molecular variance (AMOVA) was conducted to apportion the variations among and within the S. muticum populations. It indicated that variations among populations were higher than those within populations, being 55.82% verse 44.18% by RAPD and 55.21% verse 44.79% by ISSR, respectively. Furthermore, the Mantel test suggested that genetic differentiations among populations were related to the geographical distances (r > 0.6), namely, conformed to the IBD (isolation by distance) model, as expected from UPGMA (unweighted pair group method with arithmetic averages) cluster analysis. On the whole, the high genetic structuring among the four S. muticum populations along the distant locations was clearly indicated in RAPD and ISSR analyses (r > 0.9, P < 0.05) in our study.

Population genetic structure of Sargassum thunbergii (Fucales, Phaeophyta) detected by RAPD and ISSR markers

Genetic variation of four populations of Sargassum thunbergii (Mert.) O. Kuntze and one outgroup of S. fusiforme (Harv.) Setchell from Shandong peninsula of China was studied with random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. A total of 28 RAPD primers and 19 ISSR primers were amplified, showing 174 loci and 125 loci, respectively. Calculation of genetic diversity with different indicators (P%, percentage of polymorphic loci; H, the expected heterozygosity; I, Shannon's information index) revealed low or moderate levels of genetic variations within each S. thunbergii population. High genetic differentiations were determined with pairwise Nei's unbiased genetic distance (D) and fixation index (F-ST) between the populations. The Mantel test showed that two types of matrices of D and FST were highly correlated, whether from RAPD or ISSR data, r=0.9310 (P = 0.008) and 0.9313 (P=0.009) respectively. Analysis of molecular variance (AMOVA) was used to apportion the variations between and within the S. thunbergii populations. It indicated that the variations among populations were higher than those within populations, being 57.57% versus 42.43% by RAPD and 59.52% versus 40.08% by ISSR, respectively. Furthermore, the Mantel test suggested that the genetic differentiations between the four populations were related to the geographical distances (r > 0.5), i.e., they conformed to the IBD (isolation by distance) model, as expected from UPGMA (unweighted pair group method with arithmetic averages) cluster analysis. As a whole, the high genetic structuring between the four S. thunbergii populations along distant locations was clearly indicated in the RAPD and ISSR analyses (r > 0.8) in our study.

Characterizing a monotopic membrane enzyme. Biochemical, enzymatic and crystallization studies on Aquifex aeolicus sulfide:quinone oxidoreductase

Monotopic membrane proteins are membrane proteins that interact with only one leaflet of the lipid bilayer and do not possess transmembrane spanning segments. They are endowed with important physiological functions but until now only few of them have been studied. Here we present a detailed biochemical, enzymatic and crystallographic characterization of the monotopic membrane protein sulfide:quinone oxidoreductase. Sulfide:quinone oxidoreductase is a ubiquitous enzyme involved in sulfide detoxification, in sulfide-dependent respiration and photosynthesis, and in heavy metal tolerance. It may also play a crucial role in mammals, including humans, because sulfide acts as a neurotransmitter in these organisms. We isolated and purified sulfide:quinone oxidoreductase from the native membranes of the hyperthermophilic bacterium Aquifex aeolicus. We studied the pure and solubilized enzyme by denaturing and non-denaturing polyacrylamide electrophoresis, size-exclusion chromatography, cross-linking, analytical ultracentrifugation, visible and ultraviolet spectroscopy, mass spectrometry and electron microscopy. Additionally, we report the characterization of its enzymatic activity before and after crystallization. Finally, we discuss the crystallization of sulfide:quinone oxidoreductase in respect to its membrane topology and we propose a classification of monotopic membrane protein crystal lattices. Our data support and complement an earlier description of the three-dimensional structure of A. aeolicus sulfide:quinone oxidoreductase (M. Marcia, U. Ermler, G. Peng, H. Michel, Proc Natl Acad Sci USA, 106 (2009) 9625-9630) and may serve as a reference for further studies on monotopic membrane proteins. (C) 2010 Elsevier B.V. All rights reserved.

Biochemical and electron microscopic characterization of the F1F0 ATP Synthase from the hyperthermophilic eubacterium Aquifex aeolicus

The F1F0 ATP synthase has been purified from the hyperthermophilic eubacterium Aquifex aeolicus and characterized. Its subunits have been identified by MALDI-mass spectrometry through peptide mass fingerprinting and MS/MS. It contains the canonical subunits alpha, beta, gamma, delta and epsilon of F-1 and subunits a and c of F-0. Two versions of the b subunit were found, which show a low sequence homology to each other. Most likely they form a heterodimer. An electron microscopic single particle analysis revealed clear structural details, including two stalks connecting F-1 and F-0. In several orientations the central stalk appears to be tilted and/or kinked. It is unclear whether there is a direct connection between the peripheral stalk and the 6 subunit. (c) 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Population genetics of Crassostrea ariakensis in Asia inferred from microsatellite markers

Crassostrea ariakensis is an important aquacultured oyster species in Asia, its native region. During the past decade, consideration was given to introducing C. ariakensis into Chesapeake Bay, in the United States, to help revive the declining native oyster industry and bolster the local ecosystem. Little is known about the ecology and biology of this species in Asia due to confusion with nomenclature and difficulty in accurately identifying the species of wild populations in their natural environment. Even less research has been done on the population genetics of native populations of C. ariakensis in Asia. We examined the magnitude and pattern of genetic differentiation among 10 wild populations of C. ariakensis from its confirmed distribution range using eight polymorphic microsatellite markers. Results showed a small but significant global theta (ST) (0.018), indicating genetic heterogeneity among populations. Eight genetically distinct populations were further distinguished based on population pairwise theta (ST) comparisons, including one in Japan, four in China, and three populations along the coast of South Korea. A significant positive association was detected between genetic and geographic distances among populations, suggesting a genetic pattern of isolation by distance. This research represents a novel observation on wild genetic population structuring in a coastal bivalve species along the coast of the northwest Pacific.

QTL Mapping for Frond Length and Width in Laminaria japonica Aresch (Laminarales, Phaeophyta) Using AFLP and SSR Markers

In Laminaria japonica Aresch breeding practice, two quantitative traits, frond length (FL) and frond width (FW), are the most important phenotypic selection index. In order to increase the breeding efficiency by integrating phenotypic selection and marker-assisted selection, the first set of QTL controlling the two traits were determined in F-2 family using amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers. Two prominent L. japonicas inbred lines, one with "broad and thin blade" characteristics and another with "long and narrow blade" characteristics, were applied in the hybridization to yield the F-2 mapping population with 92 individuals. A total of 287 AFLP markers and 11 SSR markers were used to construct a L. japonica genetic map. The yielded map was consisted of 28 linkage groups (LG) named LG1 to LG28, spanning 1,811.1 cM with an average interval of 6.7 cM and covering the 82.8% of the estimated genome 2,186.7 cM. While three genome-wide significant QTL were detected on LG1 (two QTL) and LG4 for "FL," explaining in total 42.36% of the phenotypic variance, two QTL were identified on LG3 and LG5 for the trait "FW," accounting for the total of 36.39% of the phenotypic variance. The gene action of these QTL was additive and partially dominant. The yielded linkage map and the detected QTL can provide a tool for further genetic analysis of two traits and be potential for maker-assisted selection in L. japonica breeding.

Genetic mapping of laminaria japonica and l. longissima using amplified fragment length polymorphism markers in a "two-way pseudo-testcross" strategy

With a "two-way pseudo-testcross" mapping strategy, we applied the amplified fragment length polymorphism (AFLP) markers to construct two moderate density genetic linkage maps for Laminaria. The linkage maps were generated from the 60 progenies of the F, cross family (Laminaria longissima Aresch. x L. japonica Miyabe) with twenty pairs of primer combinations. Of the 333 polymorphic loci scored in 60 progenies, 173 segregated in a 1:1 ratio, corresponding to DNA polymorphisms heterozygous in a single parent, and the other 58 loci existing in both parents followed a 3:1 Mendelian segregation ratio. Among the loci with 1:1 segregating ratios, 79 loci were ordered in 14 linkage groups (648.6 cM) of the paternal map, and 72 loci were ordered in 14 linkage groups (601.9 cM) of the maternal map. The average density of loci was approximately 1 per 8 cM. To investigate the homologies between two parental maps, we used 58 loci segregated 3:1 for further analysis, and deduced one homologous linkage group. The linkage data developed in these maps will be useful for detecting loci-controlling commercially important traits for Laminaria.

Genetic analysis of meio- and mito-gynogenetic stocks of Paralichthys olivaceus with microsatellite markers

Twenty microsatellite markers (Po1, Po13, Po33, Po35, Po42, Po48, Po56, Po89, Po91, kop6, kop7, kop8, kop12, kop15, kop18, kop21, kop22, kop23, kop26, Po-strl) were used to assess the meiogynogenetic and mitogynogenetic stocks of the left-eyed flounder, Paralichthys olivaceus, which were derived from single pair crossing. Twelve of the 20 loci utilized showed heterozygosity in the female and were mapped in relation to their centromeres in the meiogynogenetic diploid flounder. Microsatellite-centromere map distance, calculated under the assumption of complete interference, ranged from 15.8 cM for kop22 to 50 cM for Po13, Po56 and Po89. Excluding the kop22, the heterozygosities of the rest of the loci were close to 100%, suggesting the occurrence of near complete interference on the chromosome arms that carried these loci. In the mitogynogenetic diploid flounder, each individual showed exact homozygousity and the segregation profiles did not deviate from the Mendelian 1: 1 pattern. The results indicated that there was no lethal gene linked with the loci analyzed. Such high interference accounted for the high recombination rates and large map distances. The Po13 and Po56 loci, Po91 and kop18 loci, kop15 and kop21 loci are tightly linked on the same chromosome arm in pairs.

Identification and assessing the cultivars of Laminaria Lamx. (phaeophyceae) with molecular markers

Molecular markers were used to identify and assess cultivars of Laminaria Lamx. and to delineate their phylogenetic relationships. Random amplified polymorphic DNA (RAPD) analysis was used for detection. After screening, 11 primers were selected and they yielded 133 bands in all, of which approximately 99.2% were polymorphic. The genetic distances between gametophytes ranged from 0.412 to 0.956. Two clusters were formed with the unweighted pair group method with arithmetic mean (UPGMA) dendrogram based on the simple matching coefficient. All cultivars of Laminaria japonica Aresch. used for breeding in China fell into one cluster. L. japonica from Japan, L. saccharina (L.) Lam., and L. angustata Kjellm. formed the other cluster and showed higher genetic variation than L. japonica from China. Nuclear ribosomal DNA (rDNA) sequences, including internal transcribed spacers (ITS1 and ITS2) were studied and aligned. The nucleotides of the sequences ranged from 634 to 668, with a total of 692 positions including TTS1, ITS2, and the 5.8S coding region. The phylogenetic tree obtained by the neighbor-joining method favored, to some extent, the results revealed by RAPD analysis. The present study indicates that RAPD and ITS analyses could be used to identify and assess Laminaria germplasm and to distinguish some species and, even intraspecies, in Laminaria.

Biochemical compositions of two dominant bloom- forming species isolated from the Yangtze River Estuary in response to different nutrient conditions

Variations of cellular total lipid, total carbohydrate and total protein content of two dominant bloom-forming species (Skeletonema costatum and Prorocentrum donghaiense) isolated from the Yangtze River Estuary were examined under six different nutrient conditions in batch cultures. Daily samples were collected to estimate the cell growth, nutrient concentration and three biochemical compositions content during 7 days for S. costatum and the same sampling procedure was done every other day during 10 days for P. donghaiense. Results showed that for S. costatum, cellular total lipid content increased under phosphorus (P) limitation, but not for nitrogen (N) limitation; cellular carbohydrate were accumulated under both N and P limitation: cellular total protein content of low nutrient concentration treatments were significantly lower than that of high nutrient concentration treatments. For P. donghaiense, both cellular total lipid content and total carbohydrate content were greatly elevated as a result of N and P exhaustion, but cellular total protein content had no significant changes under nutrient limitation. In addition, the capability of accumulation of three biochemical constituents of P. donghaiense was much stronger than that of S. costatum. Pearson correlation showed that for both species, the biochemical composition of three constituents (lipid, carbohydrate and protein) had no significant relationship with extracellular N concentration, but had positive correlation with extracellular and intracellular P concentration. The capability of two species to accumulate cellular total lipid and carbohydrate under nutrient limitation may help them accommodate the fluctuating nutrient condition of the Yangtze River Estuary. The different responses of two species of cellular biochemical compositions content under different nutrient conditions may provide some evidence to explain the temporal characteristic of blooms Caused by two species in the Yangtze River Estuary. (C) 2008 Elsevier B.V. All rights reserved.