GENETIC MAPPING OF THE LAMINARIA JAPONICA (LAMINARALES, PHAEOPHYTA) USING AMPLIFIED FRAGMENT LENGTH POLYMORPHISM MARKERS

To establish a molecular-marker-assisted system of breeding and genetic study for Laminaria japonica Aresch., amplified fragment length polymorphism (AFLP) was used to construct a genetic linkage map of L. japonica featuring 230 progeny of F-2 cross population. Eighteen primer combinations produced 370 polymorphic loci and 215 polymorphic loci segregated in a 3:1 Mendelian segregation ratio (P <= 0.05). Of the 215 segregated loci, 142 were ordered into 27 linkage groups. The length of the linkage groups ranged from 6.7 to 90.3 centimorgans (cM) with an average length of 49.6 cM, and the total length was 1,085.8 cM, which covered 68.4% of the estimated 1,586.9 cM genome. The number of mapped markers on each linkage group ranged from 2 to 12, averaging 5.3 markers per group. The average density of the markers was 1 per 9.4 cM. Based on the marker density and the resolution of the map, the constructed linkage map can satisfy the need for quantitative trait locus (QTL) location and molecular-marker-assisted breeding for Laminaria.

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.

Isolation and mapping of telomeric pentanucleotide (TAACC)(n) repeats of the pacific whiteleg shrimp, Penaeus vannamei, using fluorescence in situ hybridization

To develop genetic and physical maps for shrimp, accurate information on the actual number of chromosomes and a large number of genetic markers is needed. Previous reports have shown two different chromosome numbers for the Pacific whiteleg shrimp, Penaeus vannamei, the most important penaeid shrimp species cultured in the Western hemisphere. Preliminary results obtained by direct sequencing of clones from a Sau3A-digested genomic library of P. vannamei ovary identified a large number of (TAACC/GGTTA)-containing SSRs. The objectives of this study were to (1) examine the frequency of (TAACC)(n) repeats in 662 P. vannamei genomic clones that were directly sequenced, and perform homology searches of these clones, (2) confirm the number of chromosomes in testis of P. vannamei, and (3) localize the TAACC repeats in P. vannamei chromosome spreads using fluorescence in situ hybridization (FISH). Results for objective I showed that 395 out of the 662 clones sequenced contained single or multiple SSRs with three or more repeat motifs, 199 of which contained variable tandem repeats of the pentanucleotide (TAACC/GGTTA),, with 3 to 14 copies per sequence. The frequency of (TAACC)n repeats in P. vannamei is 4.68 kb for SSRs with five or more repeat motifs. Sequence comparisons using the BLASTN nonredundant and expressed sequence tag (EST) databases indicated that most of the TAACC-containing clones were similar to either the core pentanucleotide repeat in PVPENTREP locus (GenBank accession no. X82619) or portions of 28S rRNA. Transposable elements (transposase for Tn1000 and reverse transcriptase family members), hypothetical or unnamed protein products, and genes of known function such as 18S and 28S rRNAs, heat shock protein 70, and thrombospondin were identified in non-TAACC-containing clones. For objective 2, the meiotic chromosome number of P. vannamei was confirmed as N = 44. For objective 3, four FISH probes (P1 to P4) containing different numbers of TAACC repeats produced positive signals on telomeres of P. vannamei chromosomes. A few chromosomes had positive signals interstitially. Probe signal strength and chromosome coverage differed in the general order of P1 > P2 > P3 > P4, which correlated with the length of TAACC repeats within the probes: 83, 66, 35, and 30 bp, respectively, suggesting that the TAACC repeats, and not the flanking sequences, produced the TAACC signals at chromosome ends and TAACC is likely the telomere sequence for P. vannamei.

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.

Characterization and mapping of expressed sequence tags isolated from a subtracted cDNA library of Litopenaeus vannamei injected with white spot syndrome virus

A large number of polymorphic simple sequence repeats (SSRs) or microsatellites are needed to develop a genetic map for shrimp. However, developing an SSR map is very time-consuming, expensive, and most SSRs are not specifically linked to gene loci of immediate interest. We report here on our strategy to develop polymorphic markers using expressed sequence tags (ESTs) by designing primers flanking single or multiple SSRs with three or more repeats. A subtracted cDNA library was prepared using RNA from specific pathogen-free (SPF) Litopenaeus vannamei juveniles (similar to 1 g) collected before (0) and after (48 h) inoculation with the China isolate of white spot syndrome virus (WSSV). A total of 224 clones were sequenced, 194 of which were useful for homology comparisons against annotated genes in NCBI nonredundant (nr) and protein databases, providing 179 sequences encoded by nuclear DNA, 4 mitochondrial DNA, and 11 were similar to portions of WSSV genome. The nuclear sequences clustered in 43 groups, 11 of which were homologous to various ESTs of unknown function, 4 had no homology to any sequence, and 28 showed similarities to known genes of invertebrates and vertebrates, representatives of cellular metabolic processes such as calcium ion balance, cytoskeleton mRNAs, and protein synthesis. A few sequences were homologous to immune system-related (allergens) genes and two were similar to motifs of the sex-lethal gene of Drosophila. A large number of EST sequences were similar to domains of the EF-hand superfamily (Ca2+ binding motif and FRQ protein domain of myosin light chains). Single or multiple SSRs with three or more repeats were found in approximately 61 % of the 179 nuclear sequences. Primer sets were designed from 28 sequences representing 19 known or putative genes and tested for polymorphism (EST-SSR marker) in a small test panel containing 16 individuals. Ten (53%) of the 19 putative or unknown function genes were polymorphic, 4 monomorphic, and 3 either failed to satisfactorily amplify genomic DNA or the allele amplification conditions need to be further optimized. Five polymorphic ESTs were genotyped with the entire reference mapping family, two of them (actin, accession #CX535973 and shrimp allergen arginine kinase, accession #CX535999) did not amplify with all offspring of the IRMF panel suggesting presence of null alleles, and three of them amplified in most of the IRM F offspring and were used for linkage analysis. EF-hand motif of myosin light chain (accession #CX535935) was placed in ShrimpMap's linkage group 7, whereas ribosomal protein S5 (accession #CX535957) and troponin I (accession #CX535976) remained unassigned. Results indicate that (a) a large number of ESTs isolated from this cDNA library are similar to cytoskeleton mRNAs and may reflect a normal pathway of the cellular response after im infection with WSSV, and (b) primers flanking single or multiple SSRs with three or more repeats from shrimp ESTs could be an efficient approach to develop polymorphic markers useful for linkage mapping. Work is underway to map additional SSR-containing ESTs from this and other cDNA libraries as a plausible strategy to increase marker density in ShrimpMap.

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.

Chromosomal mapping of 5S ribosomal RNA genes in the eastern oyster, Crassostrea virginica gmelin by fluorescence in situ hybridization

Chromosomal location of the 5S ribosomal RNA gene was studied in the eastern oyster, Crassostrea virginica Gmelin. using fluorescence in situ hybridization (FISH). Metaphase chromosomes were obtained from early embryos, and the FISH probe was made by PCR (polymerase chain reaction) amplification of the 5S rRNA gene and labeled by incorporation of digoxigenin-1 1-dUTP during PCR. Hybridization was detected with fluorescein-labeled antidigoxigenin antibodies. Two pairs of FISH signals were observed on metaphase chromosomes. Karyotypic analysis showed that the 5S rRNA gene cluster is interstitially located on short arms of chromosomes 5 and 6. On chromosome 5, the 5S rRNA genes were located immediately next to the centromere, whereas on chromosome 6, they were located approximately half way between the telomere and the centromere. Chromosomes of C. virginica are difficult to identify because of their similarities in size and arm ratio, and the chromosomal location of 5S rRNA genes provides unambiguous identification of chromosomes 5 and 6. Previous studies have mapped the major rRNA gene cluster (18S-5.8S-28S) to chromosome 2. and this study shows that the 5S rRNA gene cluster is not linked to the major rRNA genes and duplicated during evolution.

Genetic mapping of size-related quantitative trait loci (QTL) in the bay scallop (Argopecten irradians) using AFLP and microsatellite markers

We constructed genetic linkage maps for the bay scallop Argopecten irradians using AFLP and microsatellite markers and conducted composite interval mapping (CIM) of body-size-related traits. Three hundred seventeen AFLP and 10 microsatellite markers were used for map construction. The female parent map contained 120 markers in 15 linkage groups, spanning 479.6 cM with an average interval of 7.0 cM. The male parent map had 190 markers in 17 linkage groups, covering 883.8 cM at 7.2 cM per marker. The observed coverage was 70.4% for the female and 81.1% for the male map. Markers that were distorted toward the same direction were closely linked to each other on the genetic maps, suggesting the presence of genes important for survival. Six size-related traits, shell length, shell height, shell width, total weight, soft tissue weight, and shell weight, were measured for QTL mapping. The size data were significantly correlated with each other. We subjected the data, log transformed firstly, to a principle component analysis and use the first principle component for QTL mapping. CIM analysis revealed one significant QTL (LOD=2.69, 1000 permutation, P<0.05) in linkage group 3 on the female parent map. The mapping of size-related QTL in this study raises the possibility of improving the growth of bay scallops through marker-assisted selection. (c) 2007 Published by Elsevier B.V.

Identification and mapping of amplified fragment length polymorphism markers linked to shell color in bay scallop, Argopecten irradians irradians (Lamarck, 1819)

Amplified fragment length polymorphisms (AFLP) were used to study the inheritance of shell color in Argopecten irradians. Two scallops, one with orange and the other with white shells, were used as parents to produce four F-1 families by selfing and outcrossing. Eighty-eight progeny, 37 orange and 51 white, were randomly selected from one of the families for segregation and mapping analysis with AFLP and microsatellite markers. Twenty-five AFLP primer pairs were screened, yielding 1138 fragments, among which 148 (13.0%) were polymorphic in two parents and segregated in progeny. Six AFLP markers showed significant (P < 0.05) association with shell color. All six loci were mapped to one linkage group. One of the markers, F1f335, is completely linked to the gene for orange shell, which we designated as Orange1, without any recombination in the progeny we sampled. The marker was amplified in the orange parent and all orange progeny, but absent in the white parent and all the white progeny. The close linkage between F1f335 and Orange1 was validated using bulk segregation analysis in two natural populations, and all our data indicate that F1f335 is specific for the shell color gene, Orange1. The genomic mapping of a shell color gene in bay scallop improves our understanding of shell color inheritance and may contribute to the breeding of molluscs with desired shell colors.

Chromosomal mapping of major ribosomal rRNA genes in the hard clam (Mercenaria mercenaria) using fluorescence in situ hybridization

Karyotype and chromosomal location of the major ribosomal RNA genes were studied in the hard clam (Mercenaria mercenaria Linnaeus) using fluorescence in situ hybridization (FISH). Metaphase chromosomes were obtained from early embryos. Internal transcribed spacers (ITS) between major RNA genes were amplified and used as FISH probes. The probes were labeled with digoxigenin-11-dUTP by polymerase chain reaction and detected with fluorescein-labeled anti-digoxigenin antibodies. FISH with the ITS probes produced two to four signals per nucleus or metaphase. M. mercenaria had a haploid number of 19 chromosomes with a karyotype of seven metacentric, four metacentric or submetacentric, seven submetacentric, and one submetacentric or subtelocentric chromosomes (7M + 4M/SM + 7SM + 1SM/ST). Two ITS loci were observed: one located near the centromere on the long arm of Chromosome 10 and the other at the telomere of the short arm of Chromosome 12. FISH signals on Chromosome 10 are strong and consistent, while signals on Chromosome 12 are variable. This study provides the first karyotype and chromosomal assignment of the major RNA genes in M. mercenaria. Similar studies in a wide range of species are needed to understand the role of chromosomal changes in bivalve evolution.

Chromosomal mapping of the major ribosomal RNA genes in the dwarf surfclam (Mulinia lateralis Say)

Chromosomal location of the major ribosomal RNA genes (rRNA) were studied in the dwarf surfclam (Mulinia lateralis, Say) using fluorescence in situ hybridization (FISH). FISH probes for the rRNA genes were made by polymerase chain reaction (PCR), labeled with digoxigenin-11-dUTP and detected with fluorescein-labeled antidigoxigenin antibodies. Mulinia lateralis had a diploid number of 38 chromosomes and all chromosomes were telocentric. FISH with the rRNA probe produced positive and consistent signals on two pairs of chromosomes: Chromosome 15 with a relative length of 4.6% and Chromosome 19, the shortest chromosome. Both loci were telomeric. The rRNA location provides the first physical landmark of the M. lateralis genome.

Assessing the potential of VEGETATION sensor data for mapping snow and ice cover: a Normalized Difference Snow and Ice Index

The VEGETATION (VGT) sensor in SPOT 4 has four spectral bands that are equivalent to Landsat Thematic Mapper (TM) bands (blue, red, near-infrared and mid-infrared spectral bands) and provides daily images of the global land surface at a 1-km spatial resolution. We propose a new index for identifying and mapping of snow ice cover, namely the Normalized Difference Snow/Ice Index (NDSII), which uses reflectance values of red and mid-infrared spectral bands of Landsat TM and VGT. For Landsat TM data, NDSII is calculated as NDSIITM =(TM3 -TM5)/(TM3 +TM5); for VGT data, NDSII is calculated as NDSIIVGT =(B2- MIR)/(B2 + MIR). As a case study we used a Landsat TM image that covers the eastern part of the Qilian mountain range in the Qinghai-Xizang (Tibetan) plateau of China. NDSIITM gave similar estimates of the area and spatial distribution of snow/ice cover to the Normalized Difference Snow Index (NDSI=(TM2-TM5)/(TM2+TM5)) which has been proposed by Hall et al. The results indicated that the VGT sensor might have the potential for operational monitoring and mapping of snow/ice cover from regional to global scales, when using NDSIIVGT.

汉字形音义加工时间进程的事件相关电位（ERP）研究

The time-courses of orthographic, phonological and semantic processing of Chinese characters were investigated systematically with multi-channel event-related potentials (ERPs). New evidences concerning whether phonology or semantics is processed first and whether phonology mediates semantic access were obtained, supporting and developing the new concept of repetition, overlapping, and alternating processing in Chinese character recognition. Statistic parameter mapping based on physiological double dissociation has been developed. Seven experiments were conducted: I) deciding which type of structure, left-right or non-left-right, the character displayed on the screen was; 2) deciding whether or not there was a vowel/a/in the pronunciation of the character; 3) deciding which classification, natural object or non-natural object, the character was; 4) deciding which color, red or green, the character was; 5) deciding which color, red or green, the non-character was; 6) fixing on the non-character; 7) fixing on the crosslet. The main results are: 1. N240 and P240:N240 and P240 localized at occipital and prefrontal respectively were found in experiments 1, 2, 3, and 4, but not in experiments 5, 6, or 7. The difference between the former 4 and the latter 3 experiments was only their stimuli: the former's were true Chinese characters while the latter's were non-characters or crosslet. Thus Chinese characters were related to these two components, which reflected unique processing of Chinese characters peaking at about 240 msec. 2. Basic visual feature analysis: In comparison with experiment 7 there was a common cognitive process in experiments 1, 2, 4, and 6 - basic visual feature analysis. The corresponding ERP amplitude increase in most sites started from about 60 msec. 3. Orthography: The ERP differences located at the main processing area of orthography (occipital) between experiments 1, 2, 3, 4 and experiment 5 started from about 130 msec. This was the category difference between Chinese characters and non-characters, which revealed that orthographic processing started from about 130 msec. The ERP differences between the experiments 1, 2, 3 and the experiment 4 occurred in 210-250, 230-240, and 190-250 msec respectively, suggesting orthography was processed again. These were the differences between language and non-language tasks, which revealed a higher level processing than that in the above mentioned 130 msec. All the phenomena imply that the orthographic processing does not finished in one time of processing; the second time of processing is not a simple repetition, but a higher level one. 4. Phonology: The ERPs of experiment 2 (phonological task) were significantly stronger than those of experiment 3 (semantic task) at the main processing areas of phonology (temporal and left prefrontal) starting from about 270 msec, which revealed phonologic processing. The ERP differences at left frontal between experiment 2 and experiment 1 (orthographic task) started from about 250 msec. When comparing phonological task with experiment 4 (character color decision), the ERP differences at left temporal and prefrontal started from about 220 msec. Thus phonological processing may start before 220 msec. 5. Semantic: The ERPs of experiment 3 (semantic task) were significantly stronger than those of experiment 2 (phonological task) at the main processing areas of semantics (parietal and occipital) starting from about 290 msec, which revealed semantic processing. The ERP differences at these areas between experiment 3 and experiment 4 (character color decision) started from about 270 msec. The ERP differences between experiment 3 and experiment 1 (orthographic task) started from about 260 msec. Thus semantic processing may start before 260 msec. 6. Overlapping of phonological and semantic processing: From about 270 to 350 msec, the ERPs of experiment 2 (phonological task) were significantly larger than those of experiment 3 (semantic task) at the main processing areas of phonology (temporal and left prefrontal); while from about 290-360 msec, the ERPs of experiment 3 were significantly larger than those of experiment 2 at the main processing areas of semantics (frontal, parietal, and occipital). Thus phonological processing may start earlier than semantic and their time-courses may alternate, which reveals parallel processing. 7. Semantic processing needs part phonology: When experiment 1 (orthographic task) served as baseline, the ERPs of experiment 2 and 3 (phonological and semantic tasks) significantly increased at the main processing areas of phonology (left temporal and frontal) starting from about 250 msec. The ERPs of experiment 3, besides, increased significantly at the main processing areas of semantics (parietal and frontal) starting from about 260 msec. When experiment 4 (character color decision) served as baseline, the ERPs of experiment 2 and 3 significantly increased at phonological areas (left temporal and frontal) starting from about 220 msec. The ERPs of experiment 3, similarly, increased significantly at semantic areas (parietal and frontal) starting from about270 msec. Hence, before semantic processing, a part of phonological information may be required. The conclusion could be got from above results in the present experimental conditions: 1. The basic visual feature processing starts from about 60 msec; 2. Orthographic processing starts from about 130 msec, and repeats at about 240 msec. The second processing is not simple repetition of the first one, but a higher level processing; 3. Phonological processing begins earlier than semantic, and their time-courses overlap; 4. Before semantic processing, a part of phonological information may be required; 5. The repetition, overlapping, and alternating of the orthographic, phonological and semantic processing of Chinese characters could exist in cognition. Thus the problem of whether phonology mediates semantics access is not a simple, but a complicated issue.