AFLP-based genetic linkage maps of the Pacific oyster Crassostrea gigas Thunberg

Amplified fragment length polymorphisms (AFLPs) were used for genome mapping in the Pacific Oyster Crassostrea gigas Thunberg. Seventeen selected primer combinations produced 1106 peaks, of which 384 (34.7%) were polymorphic in a backcross family. Among the polymorphic markers, 349 were segregating through either the female or the male parent. Chi-square analysis indicated that 255 (73.1%) of the markers segregated in a Mendelian ratio, and 94 (26.9%) showed significant (P < 0.05) segregation distortion. Separate genetic linkage maps were constructed for the female and male parents. The female framework map consisted of 119 markers in 11 linkage groups, spanning 1030.7 cM, with an average interval of 9.5 cM per marker. The male map contained 96 markers in 10 linkage groups, covering 758.4 cM, with 8.8 cM per marker. The estimated genome length of the Pacific oyster was 1258 cM for the female and 933 cM for the male, and the observed coverage was 82.0% for the female map and 81.3% for the male map. Most distorted markers were deficient for homozygotes and closely linked to each other on the genetic map, suggesting the presence of major recessive deleterious genes in the Pacific oyster.

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.

A preliminary genetic linkage map of the pacific abalone Haliotis discus hannai Ino

Preliminary genetic linkage maps were constructed for the Pacific abalone (Haliotis discus hannai Ino) using amplified fragment length polymorphism (AFLP), randomly amplified polymorphic DNA (RAPD), and microsatellite markers segregating in a F, family. Nine microsatellite loci, 41 RAPD, and 2688 AFLP markers were genotyped in the parents and 86 progeny of the mapping family. Among the 2738 markers, 384 (including 365 AFLP markers, 10 RAPD markers, and 9 microsatellite loci) were polymorphic and segregated in one or both parents: 241 in the female and 146 in the male. The majority of these markers, 232 in the female and 134 in the male, segregated according to the expected 1:1 Mendelian ratio (alpha = 0.05). Two genetic linkage maps were constructed using markers segregating in the female or the male parent. The female framework map consisted of 119 markers in 22 linkage groups, covering 1773.6 cM with an average intermarker space of 18.3 cM. The male framework map contained 94 markers in 19 linkage groups, spanning 1365.9 cM with an average intermarker space of 18.2 cM. The sex determination locus was mapped to the male map but not to the female map, suggesting a XY-male determination mechanism. Distorted markers showing excess of homozygotes were mapped in clusters, probably because of their linkage to a gene that is incompatible between two parental populations.

Its length polymorphism in oysters and its use in species identification

In an effort to develop genetic markers for oyster identification, we studied length polymorphism in internal transcribed spacers (ITS) between major ribosomal RNA genes in 12 common species of Ostreidae: Crassostrea virginica, C. rhizophorae, C. gigas, C. angulata, C. sikamea, C. ariakensis, C. hongkongensis, Saccostrea echinata, S. glomerata, Ostrea angasi, O. edulis, and O. conchaphila. We designed two pairs of primers and optimized PCR conditions for simultaneous amplification of ITS 1 and ITS2 in a single PCR. Amplification was successful in all 12 species, and PCR products were visualized on high-resolution agarose gels. ITS2 was longer than ITS 1 in all Crassostrea and Saccostrea species, whereas they were about the same size in the three Ostrea species. No intraspecific variation in ITS length was detected. Among species, the length of ITS I and ITS2 was polymorphic and provided unique identification of 8 species or species pairs: C. ariakensis, C. hongkongensis, C. sikamea, O. conchaphila, C. virginica/C. rhizophorae, C. gigas/C. angulata, S. echinata/S. glonzerata, and O. angasi/O. edulis. The ITS assay provides simple, rapid and effective identification of C. ariakensis and several other oyster species. Because the primer sequences are conserved, the ITS assay may be useful in the identification of other bivalve species.

Cytological and Molecular Identification of Alien Chromatin in Giant Spike Wheat Germplasm

Alien chromosomes of twelve giant spike wheat germplasm lines were identified by C-banding, genomic in situ hybridization (GISH), sequence characterized amplified region (SCAR), and random amplified polymorphic DNA (RAPD). All lines showed a chromosome number of 2n = 42, five of them carried both a pair of wheat-rye (Triticum aestivum-Secale cereal) 1BL/1RS translocation chromosomes and a pair of Agropyron intermedium (Ai) chromosomes, three carried a pair of Ai chromosomes only, three others carried a pair of 1BL/1RS chromosomes only, and one carried neither 1BL/1BS nor Ai chromosome. Further identification revealed that the identical Ai chromosome in these germplasm lines substituted the chromosome 2D of common wheat (Triticum aestivum L.), designated as 2Ai. The genetic implication and further utilization of 2Ai in wheat improvement were also discussed.

Isolation and characterization of microsatellite DNA primers in Juniperus przewalskii Kom (Cupressaceae)

Juniperus przewalskii (Cupressaceae) is a dominant tree species endemic to the northeast Qinghai-Tibetan Plateau. This species plays an important role in maintaining the arid ecosystem in this region. However, natural distributions of this species have been declined. In order to develop effective conservation methods, it is important to know the distribution of the genetic diversity within and among populations. In this study, we developed nine new microsatellite loci for this species. We used the combining biotin capture method to enrich AG/CT/AC/GGT microsatellites. The polymorphisms of each locus were further assessed in 12 individuals from four geographically distant populations. The number of alleles per locus varied from three to six and expected heterozygosity ranged from 0.58 to 0.70. These loci together provide a useful tool to investigate the genetic diversity of this species. In addition, all markers have been crossly checked in the other four congeneric species.

Development of 10 microsatellite loci for Rheum tanguticum (Polygonaceae)

Rhubarb is an important Traditional Chinese Medicine. However, the wild resource has been declining. In order to design appropriate conservation methods for the official species across their natural distributions, it is important to characterize their genetic diversity. Here, we describe the development of 10 new microsatellite loci for AC/TG/CCA in Rheum tanguticum. The microsatellites were enriched using the combined biotin capture method. The polymorphism of each locus was further assessed in 12 individuals from four geographically distinct populations of this species. The number of alleles ranged from three to seven and the expected heterozygosity ranged from 0.53 to 0.73. All markers have been checked in the other three species in the genus and two of them together comprise the official medicinal rhubarb resource, with R. tanguticum. These microsatellite markers could. provide a useful tool for genetic and conservation studies of the rhubarb species.

Genetic variation in the endangered Anisodus tanguticus (Solanaceae), an alpine perennial endemic to the Qinghai-Tibetan Plateau

We used random amplified polymorphic DNA markers (RAPDs) to assess genetic variation between- and within-populations of Anisodus tanguticus (Solanaceae), an endangered perennial endemic to the Qinghai-Tibetan Plateau with important medicinal value. We recorded a total of 92 amplified bands, using 12 RAPD primers, 76 of which (P = 82.61%) were polymorphic, and calculated values of H-t and H-sp of 0.3015 and 0.4459, respectively, suggesting a remarkably high rate of genetic variation at the species level. The average within-population diversity also appeared to be high, with P, H-e and H-pop values of 55.11%, 0.1948 and 0.2918, respectively. Analyses of molecular variance (AMOVA) showed that among- and between-population genetic variation accounted for 67.02% and 32.98% of the total genetic variation, respectively. In addition, Nei's coefficient of differentiation (G(ST)) was found to be high (0.35), confirming the relatively high level of genetic differentiation among the populations. These differentiation coefficients are higher than mean corresponding coefficients for outbreeding species, but lower than reported coefficients for some rare species from this region. The genetic structure of A. tanguticus has probably been shaped by its breeding attributes, biogeographic history and human impact due to collection for medicinal purposes. The observed genetic variations suggest that as many populations as possible should be considered in any planned in situ or ex situ conservation programs for this species.

Genetic variation and phylogeographic history of Picea likiangensis revealed by RAPD markers

Repeated cycles of retreat and recolonization during the Quaternary ice ages are thought to have greatly influenced current species distributions and their genetic diversity. It remains unclear how this climatic oscillation has affected the distribution of genetic diversity between populations of wind-pollinated conifers in the Qinghai-Tibetan region. In this study, we investigated the within-species genetic diversity and phylogenetic relationships of Picea likiangensis, a dominant forest species in this region using polymorphic DNA (RAPD) markers. Our results suggest that this species has high overall genetic diversity, with 85.42% of loci being polymorphic and an average expected heterozygosity (H (E)) of 0.239. However, there were relatively low levels of polymorphism at population levels and the differences between populations were not significant, with percentages of polymorphic bands (PPB) ranging from 46.88 to 69.76%, Nei's gene diversity (H (E)) from 0.179 to 0.289 and Shannon's indices (Hpop) from 0.267 to 0.421. In accordance with our proposed hypothesis, a high level of genetic differentiation among populations was detected based on Nei's genetic diversity (G (ST) = 0.256) and AMOVA analysis (Phi (st) = 0.236). Gene flow between populations was found to be limited (Nm = 1.4532) and far lower than reported for other conifer species with wide distribution ranges from other regions. No clusters corresponding to three morphological varieties found in the south, north and west, respectively, were detected in either UPGMA or PCO analyses. Our results suggest that this species may have had different refugia during the glacial stages in the southern region and that the northern variety may have multiple origins from these different refugia.

ISSR analysis of genetic diversity of the Qinghai-Tibet Plateau endemic, Rhodiola chrysanthemifolia (Crassulaceae)

Inter-simple sequence repeat markers (ISSR) were used to estimate genetic diversity within and among 10 populations of Rhodiola chrysanthemifolia along Nianqingtangula Mountains and Brahmaputra, a species endemic to the Qinghai-Tibet Plateau and an endangered medicinal plant. Of the 100 primers screened, 13 produced highly polymorphic DNA fragments. Using these primers, 116 discernible DNA fragments were generated of which 104 (89.7%) were polymorphic, indicating substantial genetic diversity at the species level. Genetic diversity measured by the percentage of polymorphic bands (PPB) at the population level ranged from 21.97% to 48.8%. Analysis of molecular variance (AMOVA) showed that the genetic variation was found mainly among populations (77.3%), but no regional differentiation was discernible. Variance within populations was only 22.7%. The main factor responsible for this high level of differentiation among populations is probably the historical geographical and genetic isolation of populations in a harsh mountainous environment. Concerning the management of R. chrysanthemifolia, the high genetic differentiation of populations indicates the necessity of conserving the maximum possible number of populations. (c) 2006 Elsevier Ltd. All rights reserved.

Patterns of genetic variation in Swertia przewalskii, an endangered endemic species of the Qinghai-Tibet Plateau

Swertia przewalskii Pissjauk. (Gentianaceae) is a critically endangered and endemic plant of the Qinghai-Tibet Plateau in China. RAPD and ISSR analyses were carried out on a total of 63 individuals to assess the extent of genetic variation in the remaining three populations. Percentage of polymorphic bands was 94% (156 bands) for RAPD and 96% (222 bands) for ISSR. A pairwise distance measure calculated from the RAPD and ISSR data was used as input for analysis of molecular variance (AMOVA). AMOVA indicated that a high proportion of the total genetic variation (52% for RAPD and 56% for ISSR) was found among populations; pairwise Phi(ST) comparisons showed that the three populations examined were significantly different (p < 0.001). Significant genetic differentiation was found based on different measures (AMOVA and Hickory theta(B)) in S. przewalskii (0.52 on RAPD and 0.56 on ISSR; 0.46 on RAPD and 0.45 on ISSR). The differentiation of the populations corresponded to low average gene flow (0.28 based on RAPD and 0.31 based on ISSR), whereas genetic distance-based clustering and coalescent-based assignment analyses revealed significant genetic isolation among populations. Our results indicate that genetic diversity is independent of population size. We conclude that although sexual reproduction and gene flow between populations of S. przewalskii are very limited, they have preserved high levels of genetic diversity. The main factors responsible for the high level of difference among populations are the isolation and recent fragmentation under human disturbance.

Geographic variation in the leaf essential oils of Juniperus sabina L. and J-sablina var. arenaria (E.H. Wilson) Farjon

The composition of the leaf oils from seven populations of J. sabina L., one population of Juniperus sabina var. arenaria (E. H. Wilson) Farjon were examined for their geographic variation. In addition, the leaf oils of J. chinensis L. and J. davurica Pall. were compared to J. sabina. Juniperus sabina var. arenarla, the sand loving juniper, oil was found to be very similar to that of J. davurica, Mongolia, and J. sabina, on sand dunes in Mongolia. This suggests that J. sabina var. arenaria might be conspecific with J. davurica. Farjon's move (2001) of J. sabina var. arenaria out of J. chinensis is supported. Considerable differentiation was found in populations of J. sabina from the Iberian peninsula. Cedrol, citronellol, safrole, trans-sabinyl acetate, terpinen-4-ol and beta-thujone were found to be polymorphic in several populations.

Genetic variation within and among populations of Rhodiola alsia (Crassulaceae) native to the Tibetan Plateau as detected by ISSR markers

Genetic variation of 10 Rhodiola alsia ( Crassulaceae) populations from the Qinghai - Tibet Plateau of China was investigated using intersimple sequence repeat (ISSR) markers. R. alsia is an endemic species of the Qinghai - Tibet Plateau. Of the 100 primers screened, 13 were highly polymorphic. Using these primers, 140 discernible DNA fragments were generated with 112 (80%) being polymorphic, indicating pronounced genetic variation at the species level. Also there were high levels of polymorphism at the population level with the percentage of polymorphic bands (PPB) ranging from 63.4 to 88.6%. Analysis of molecular variance (AMOVA) showed that the genetic variation was mainly found among populations (70.3%) and variance within populations was 29.7%. The main factors responsible for the high level of differentiation among populations are probably the isolation from other populations and clonal propagation of this species. Occasional sexual reproduction might occur in order to maintain high levels of variation within populations. Environmental conditions could also influence population genetic structure as they occur in severe habitats. The strong genetic differentiation among populations in our study indicates that the conservation of genetic variability in R. alsia requires maintenance of as many populations as possible.

RAPD profiling in detecting genetic variation in endemic Coelonema (Brassicaceae) of Qinghai-Tibet Plateau of China

Random amplified polymorphic DNA ( RAPD) markers were used to measure genetic diversity of Coelonema draboides ( Brassicaceae), a genus endemic to the Qilian Mountains of the Qinghai-Tibet Plateau. We sampled 90 individuals in 30 populations of Coelonema draboides from Datong and Huzhu counties of Qinghai Province in P. R. China. A total of 186 amplified bands were scored from the 14 RAPD primers, with a mean of 13.3 amplified bands per primer, and 87% ( 161 bands) polymorphic bands (PPB) was found. Analysis of molecular variance (AMOVA) shows that a large proportion of genetic variation (84.2%) resides among individuals within populations, while only 15.8% resides among populations. The species shows higher genetic diversity between individuals than other endemic and endangered plants. The RAPDs provide a useful tool for assessing genetic diversity of rare, endemic species and for resolving relationships among populations. The results show that the genetic diversity of this species is high, possibly allowing it to adapt more easily to environmental variations. The main factor responsible for the high level of differentiation within populations and the low level of diversity among populations is probably the outcrossing and long-lived nature of this species. Some long-distance dispersal, even among far separated populations, is also a crucial determinant for the pattern of genetic variation in the species. This distributive pattern of genetic variation of C. draboides populations provides important baseline data for conservation and collection strategies for the species. It is suggested that only populations in different habitats should be studied and protected, not all populations, so as to retain as much genetic diversity as possible.