Development of polymorphic EST markers in Penaeus monodon: applications in penaeid genetics

The objective of this study was to develop type I markers for genome mapping and other genetic studies of Penaeus monodon. Primers were designed based on expressed sequence tags (ESTs) from a P monodon cephalothorax cDNA library to amplify 100-300 bp products. 34 of the primer pairs successfully amplified PCR products from genomic DNA. Single-strand conformation polymorphism analysis showed that similar to 30% of the ESTs tested exhibit polymorphism in a test panel of P monodon individuals. Mendelian inheritance of the EST-derived markers has been established in two international reference mapping families of P monodon, and mapping of these markers is in progress. Some ESTs were successfully amplified from other Penaeus species (P. chinensis, P japonicus and P vannamei), indicating that the markers are applicable in cross-species comparison. Two populations of P. japonicus could be differentiated using one of the ESTS. In conclusion, the polymorphic EST markers developed in this study are applicable in genome mapping and population genetic studies of penaeid shrimp. (C) 2002 Elsevier Science B.V. All rights reserved.

Genetic diversity among different clones of the gynogenetic silver crucian carp, Carassius auratus gibelio, revealed by transferrin and isozyme markers

Genetic diversity among four clones (A, D, E, F) of gynogenetic silver crucian carp was studied using transferrin and isozymes in the blood as markers. Of the five proteins investigated, three (transferrin, esterase and superoxide dismutase) indicated polymorphism and eight polymorphic loci were detected. These loci were probably encoded by codominant alleles and their inheritance patterns were analyzed. Intraclonal homogeneity and interclonal heterogeneity were observed in these clones, which allowed us to infer the clonal nature and evolutionary relationship between them. Clonal diversity in this population of silver crucian carp in China was also compared with data reported from gynogenetic crucian carp in Germany.

A linkage map of common carp (Cyprinus carpio) based on AFLP and microsatellite markers

P>Common carp (Cyprinus carpio) is an important fish for aquaculture, but genomics of this species is still in its infancy. In this study, a linkage map of common carp based on Amplified Fragment Length Polymorphism (AFLP) and microsatellite (SSR) markers has been generated using gynogenetic haploids. Of 926 markers genotyped, 151 (149 AFLPs, two SSRs) were distorted and eliminated from the linkage analyses. A total of 699 AFLP and 20 microsatellite (SSR) markers were assigned to the map, which comprised 64 linkage groups and covered 5506.9 cM Kosambi, with an average interval distance of 7.66 cM Kosambi. The normality tests on interval map distances showed a non-normal marker distribution. Visual inspection of the map distance distribution histogram showed a cluster of interval map distances on the left side of the chart, which suggested the occurrence of AFLP marker clusters. On the other hand, the lack of an obvious cluster on the right side showed that there were a few big gaps which need more markers to bridge. The correlation analysis showed a highly significant relatedness between the length of linkage group and the number of markers, indicating that the AFLP markers in this map were randomly distributed among different linkage groups. This study is helpful for research into the common carp genome and for further studies of genetics and marker-assisted breeding in this species.

R IBLL终端LA SCAR阵列探测器PM T高压控制系统的研制

介绍了R IBLL终端LA SCAR上PM T阵列电源高压控制系统。该系统以当今工程控制中常用的微型计算机作为核心器件,采用合适的外围电路达到了对PM T阵列稳定、可靠的控制,在现场测试中取得了良好的效果。

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.

Assessing genetic identity of sporophytic offspring of the brown alga Undaria pinnatifida derived from mono-crossing of gametophyte clones by use of amplified fragment length polymorphism and microsatellite markers

The haploid stage of gametophytes of the subtidal brown alga Undaria pinnatifida can be vegetatively propagated under favorable conditions. This unique characteristic makes it possible to establish independent gametophyte cell lines that are zoospore-derived. Sporophytic offspring can be generated through hybridizing the male and female gametophytes, which are derived from different cell lines. Accumulated experiences in this and other species in Laminariales demonstrated the applicability of this novel way to breed desired strains for open-sea cultivation. Sporophytic offspring originated from mono-crossing of male and female gametophyte clones were shown to have similar morphological characteristics under identical ambient conditions. However, there has been no report to relate this similarity on molecular levels. In this report, amplified fragment length polymorphism (AFLP) and microsatellite markers were used to analyze the genetic identity of sporophytic offspring of U. pinnatifida originated from two mono-crossing lines (M1 and M2), two self-breeding lines (S1 and S2) and one wild population (W). Totally 318 AFLP loci were revealed by use of 11 primer sets, of which 4.7%, 0.3%, 17.9%, 16.4% and 36.5% were polymorphic in M1, M2, S1, S2 and W, respectively. The pairwise genetic identity among the individuals of the same line was assessed. It was shown that offspring from mono-crossing lines had a higher degree of identity (95.6-100%) than self-breeding lines (87.7-98.4%) and the wild population (81.5-92.1%). Analysis by use of six microsatellite loci also revealed a higher genetic identity among individuals of the mono-crossing line, further confirming the results of AFLP analysis. Results from this investigation support, on molecular levels, the novel way to produce and maintain strains in U. pinnatifida by use of different gametophyte cell lines.

Assessment of genetic diversities of selected Laminaria (Laminariales, Phaeophyta) gametophytes by inter-simple sequence repeat analysis

Inter-simple sequence repeat (ISSR) analysis was used to assess genetic diversity among 10 pairs of male and female Laminaria gametophytes. A total of 58 amplification loci was obtained from 10 selected ISSR primers, of which 34 revealed polymorphism among the gametophytes. Genetic distances were calculated with the Dice coefficient ranging from 0.006 to 0.223. A dendrogram based on the unweighted pair-group method arithmetic (UPGMA) average showed that most male and female gametophytes of the same species were clustered together and that 10 pairs of gametophytes were divided into four groups. This was generally consistent with the taxonomic categories. The main group consisted of six pairs of gametophytes, which were selected from Laminaria japonica Aresch. by intensive inbreeding through artificial hybridization. One specific marker was cloned, but was not converted successfully into a sequence characterized amplified region (SCAR) marker. Our results demonstrate the feasibility of applying ISSR markers to evaluate Laminaria germplasm diversities.

Polymorphic EST-SSR markers and their mode of inheritance in Fenneropenaeus chinensis

229 SSRs (simple sequence repeats) were identified among 10,443 ESTs (expressed sequence tags) of Chinese shrimp (Fenneropenaeus chinensis). The average density of SSRs was one SSR per 19.1 kb of EST sequence screened. The dinucleotide repeats appeared to be the most abundant SSRs detected. Nine EST-SSR markers were detected polymorphisms of the thirty SSR primer pairs derived from F chinensis ESTs. The number of alleles per locus ranged from 5 to 15, with an average of 9.1 alleles per locus. The observed heterozygosity of nine loci ranged from 0.47 to 0.87. These loci were used successfully for pedigree analysis in three families of Fenneropenaeus chinensis. Two of the nine microsatellite loci showed the existence of null alleles. Assuming the existence of null alleles at Fc07 and Fc14 loci, the allelic inheritance mode of the EST-SSR DNA markers (Fc04, Fc06, Fc07, Fc10, Fc14, Fc18, Fc22, Fc24, and Fc27) was consistent with Mendelian segregation. (c) 2005 Elsevier B.V. All rights reserved.

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.

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.