Isolation and characterization of 17 polymorphic microsatellites in grass carp

Here we report the isolation and characterization of 17 polymorphic loci isolated from a partial genomic DNA library of grass carp (Ctenopharyngodon idellus) enriched for CA repeats. We tested variability of these microsatellites on 24 unrelated individuals collected in China. All microsatellites were polymorphic. The average allele number was 7.9 per locus, ranging from four to 13. The observed heterozygosity was from 0.46 to 0.88 with an average of 0.71, whereas the average expected heterozygosity was 0.78. Sixteen of the 17 microsatellites conformed to Hardy–Weinberg equilibrium, and inherited independently. These microsatellites can be used to study genetic diversity and population structure of wild populations, and facilitate selective breeding of cultured broodstocks.     

Isolation and characterization of microsatellites in Brassica rapa L

We report here the isolation and characterization of microsatellites, or simple sequence repeats (SSRs), in Brassica rapa. The size-fractionated genomic library was screened with (GA)(15) and (GT)(15) oligonucleotide probes. A total of 58 clones were identified as having the microsatellite repeats, and specific primer pairs were designed for 38 microsatellite loci. All primer pairs, except two, amplified fragments having the sizes expected from the sequences. Of the 36 primer pairs, 35 amplified polymorphic loci in 19 cultivars of B. rapa, while monomorphism was observed in only one primer pair. A total of 232 alleles was identified by the 36 primer pairs in 19 cultivars of B. rapa, and these primer pairs were examined also in nine Brassicaceae species. Most of the 36 primer pairs amplified the loci in the Brassicaceae species. Segregation of the microsatellites was studied in an F(2) population from a cross of doubled-haploid lines DH27 x G309. The microsatellites segregated in a co-dominant manner. These results indicate that the microsatellites isolated in this study were highly informative and could be useful tools for genetic analysis in B. rapa and other related species.     

Cellular responses of leaf explants of Cocos nucifera L. in vitro

Leaf explants of Cocos nucifera L. (coconut palm) were studied in vitro in order to establish whether or not rapid cellular changes contribute to the well known recalcitrance of coconut cells in tissue culture. Segments from the base of immature leaves were cultured on modified Eeuwens' medium at 30°C in darkness. The mitotic index, nuclear DNA amounts, cell and nuclear size were measured both before and during culture (from 0 to 70 days). There was no basipetal gradient of cell division in immature coconut leaves; the mitotic index never exceeded 2% and showed neither a positional nor temporal relationship with leaf development. Moreover the vast majority of cells were in G1 of the cell cycle. This cell cycle pattern was maintained for most of the period in culture although at 70 days there was an increase in the proportion of cells in S- and G2-phases consistent with low rates of callus formation. The nuclear: cell size ratio was constant in cells within the immature leaf irrespective of developmental age. However upon transfer to culture media, cell size but not nuclear size increased. We suggest that this uncoupling of cell and nuclear size disrupts cell co-ordination and is a key contributor to recalcitrant cellular behaviour of this species in vitro.     

Isolation and mapping of polymorphic microsatellites in cattle

Summary
A partial plasmid library with bovine genomic inserts of about 500 basepairs was screened with a (dC-dA)_n-(dG-dT)_n oligonucleotide probe for the repeated nucleotide motif (CA)_n. Eleven positive clones (0.3% of all colonies screened) were discovered and were subsequently isolated and sequenced. Eight microsatellite loci were analysed, one with eight alleles, one with seven alleles, three with six alleles, one with three alleles and two with two alleles. Six of these microsatellites were mapped by PCR-analysis of a panel of somatic hybrid lines.     

Isolation and characterization of 15 polymorphic microsatellites in the Plethodontid salamander Ensatina eschscholtzii

We developed 15 new polymorphic microsatellites for the plethodontid salamander Ensatina eschscholtzii. Loci were isolated from a genomic library from Ensatina eschscholtzii xanthoptica enriched for (AAAG)(n) repetitive elements. The number of alleles per locus ranged from 4 to 20 (mean 9) in the sampled population. Observed heterozygosity ranged from 0.37 to 1. None of the loci deviated from Hardy-Weinberg equilibrium or showed significant linkage disequilibrium after a Bonferroni correction for multiple comparisons. All loci amplified in the six other subspecies of the Ensatina eschscholtzii complex. These new markers will prove useful in measuring gene flow and population structure as well as patterns of mating and sperm use in Ensatina.     

Cloning and characterization of highly polymorphic porcine microsatellites.

A size-fractionated (200-400 bp) porcine genomic library was screened with the dinucleotide motifs (TG)n and (TC)n. The number of TG- and TC-positive clones was 83 and four, respectively, implying that the former motif is more frequent in the porcine genome, as previously reported in other species. Twenty-six TG-clones were sequenced, and the number of repeats varied between 16 and 42 with different compositions of the repetitive sequences; 17 clones had a perfect stretch of TG-repeats, four had imperfect stretches, and five had a compound structure with TG-repeats followed by TC-repeats. Primers for DNA amplification using the polymerase chain reaction (PCR) were synthesized for six loci. Ten unrelated individuals (two wild boars and eight domestic pigs of the Swedish Yorkshire breed) were screened for microsatellite polymorphism. All six microsatellite loci were polymorphic with two to seven alleles and observed heterozygosities in the range of 0.42-0.84; the inheritance of the observed polymorphism was confirmed by family studies. The characteristics of microsatellites make them highly suitable as genetic markers, and these microsatellites were isolated as a part of a pig gene mapping project.     

Isolation and characterization of highly polymorphic microsatellites in the chokka squid, Loligo reynaudii

The chokka squid, Loligo reynaudii, is the target of a commercially valuable fishery in South Africa, but little information is available on population structure and mating system. We developed 11 polymorphic microsatellite markers using both standard nonenrichment and enrichment protocols. Numbers of alleles per locus ranged from 11 to 30, and levels of expected heterozygosity ranged between 0.77 and 0.98. Three loci developed using an enriched library displayed significant departure from Hardy-Weinberg genotype proportions suggesting high frequencies of null alleles. The remaining loci should prove useful for population and parentage studies.     

Isolation and characterization of polymorphic microsatellites from Chinese mitten crab,Eriocheir sinensis

Chinese mitten crab (Eriocheir sinensis) has great commercial significance in Asia, but is an invasive organism in Europe and America. We isolated five novel microsatellites for E. sinensis. All five loci were polymorphic. The average allele number was 16.8 per locus with a range of 13 to 21, while the expected heterozygosity ranged from 0.82 to 0.91 with an average of 0.88. These markers could be applicable to studies of the genetic diversity and population structure of E. sinensis.     

Dehydration improves cryopreservation of coconut (Cocos nucifera L.)

Cryopreservation of coconut can be used as a strategy to back up the establishment of living collections which are expensive to maintain and are under constant threat from biotic and abiotic factors. Unfortunately, cryopreservation protocols still need to be developed that are capable of producing a sizeable number of field-grown plants. Therefore, we report on the development of an improved cryopreservation protocol which can be used on a wide range of coconut cultivars. The cryopreservation of zygotic embryos and their recovery to soil-growing plants was achieved through the application of four optimised steps viz.: (i) rapid dehydration; (ii) rapid cooling; (iii) rapid warming and recovery in vitro and (iv) acclimatisation and soil-supported growth. The thermal properties of water within the embryos were monitored using differential scanning calorimetry (DSC) in order to ensure that the freezable component was kept to a minimum. The feasibility of the protocol was assessed using the Malayan Yellow Dwarf (MYD) cultivar in Australia and then tested on a range of cultivars which were freshly harvested and studied in Indonesia. The most efficient protocol was one based on an 8-h rapid dehydration step followed by rapid cooling step. Best recovery percentages were obtained when a rapid warming step and an optimised in vitro culture step were used. Following this protocol, 20% (when cryopreserved 12 days after harvesting) and 40% (when cryopreserved at the time of harvest) of all MYD embryos cryopreserved could be returned to normal seedlings growing in soil. DSC showed that this protocol induced a drop in embryo fresh weight to 19% and significantly reduced the amount of water remaining that could produce ice crystals (0.1%). Of the 20 cultivars tested, 16 were found to produce between 10% and 40% normal seedlings while four cultivars generated between 0% and 10% normal seedlings after cryopreservation. This new protocol is applicable to a wide range of coconut cultivars and is useful for the routine cryopreservation of coconut genetic resources.     

Linkage mapping and QTL analysis in coconut (Cocos nucifera L.)

Different DNA marker types were used to construct linkage maps in coconut (Cocos nucifera L.; 2n = 32) for the two parents of the cross Malayan Yellow Dwarf (MYD) × Laguna Tall (LAGT). A total of 382 markers was sufficient to generate 16 linkage groups for each parent. The total genome length corresponded to 2226 cM for the LAGT map and 1266 cM for the MYD map with 4–32 markers per linkage group. Common markers allowed the association of 9 linkage groups for the two parents MYD and LAGT. QTL analysis for the trait early germination identified six loci. These QTLs correlate with early flowering and yield, representing characters which are important in coconut breeding. The co-segregation of markers with these QTLs provides the first opportunity for marker-assisted selection in coconut breeding programmes. Received: 22 September 1999 / Accepted: 29 November 1999     

Isolation and characterization of microsatellites in a perennial Apiaceae,Eryngium alpinum L.

In order to investigate the genetic structure in an endangered Alpine plant (Eryngium alpinum L.), we developed microsatellites. Two different approaches were used: an enrichment protocol and the classical technique of hybridization on nylon membranes. We identified 25 loci, 13 of which revealed to be polymorphic. The polymorphism was rather low (2–6 alleles; H_E = 0.49 ± 0.16), probably due to the short size of microsatellites (6–10 dinucleotide repeats) and to the fine spatial scale investigated. However, these markers are expected to provide a new insight about the genetic processes at work within and among E. alpinum populations.     

Endogenous cytokinins in Cocos nucifera L. in vitro cultures obtained from plumular explants

Auxin induces in vitro somatic embryogenesis in coconut plumular explants through callus formation. Embryogenic calli and non-embryogenic calli can be formed from the initial calli. Analysis of endogenous cytokinins showed the occurrence of cytokinins with aromatic and aliphatic side chains. Fourteen aliphatic cytokinins and four aromatic cytokinins were analysed in the three types of calli and all the cytokinins were found in each type, although some in larger proportions than others. The most abundant cytokinins in each type of callus were isopentenyladenine-9-glucoside, zeatin-9-glucoside, zeatin riboside, isopentenyladenine riboside, dihydrozeatin and dihydrozeatin riboside in decreasing order. Total cytokinin content was compared between the three types of calli, and it was found to be lower in embryogenic calli compared to non-embryogenic calli or initial calli. The same pattern was observed for individual cytokinins. When explants were cultured in media containing exogenously added cytokinins, the formation of embryogenic calli in the explants was reduced. When 8-azaadenine (an anticytokinin) was added the formation of embryogenic calli and somatic embryos was increased. These results suggest that the difference in somatic embryo formation capacity observed between embryogenic calli and non-embryogenic calli is related to their endogenous cytokinin contents.     

Development of Organized Structures in Callus Derived from Explants of Cocos nucifera L.

Clonal propagation of the coconut palm was attempted using tissueculture. Callus initiated from slices of seedling stem and fromthe rachillae of young inflorescences was maintained on agarmedium containing 2,4-D at 10^–4 M. Reduction of the 2,4-Dconcentration over several sub-cultures resulted in the developmentof nodular structures that resemble some of the stages in thedevelopment of the zygotic embryo. Cocos nucifera L., coconut palm, callus, organogenesis, embryogenesis, clonal propagation     

Induction and differentiation of callus from embryos of Cocos nucifera L. by IAA-conjugates

Calli from young embryos of Cocos nucifera L. were induced on B5 medium supplemented with IAA-conjugates (IAA-asp or IAA-ala) at a concentration of 2.0 mg/1 and callusing was increased by about 10% if both IAA-conjugates, IAA-asp and IAA-ala were added together. Differentiation of shoots and roots was achieved by transferring calli to B5 medium supplemented with either IAA-asp (2.0 mg/1)+Kn(2.0 mg/1) or NAA (2.0 mg/1). Complete plantlets were obtained on B5 medium supplemented with NAA (0.5 mg/1)+BAP (2.0 mg/1)+PVP (1.0 g/1).Abbreviations IAA Indole-3-acetic acid - IAA-ala Indole acetyl-L-alanine - IAA-asp Indole acetyl-L-aspartic acid - Kn Kinetin - BAP N⁶-benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - NAA -naphthalene acetic acid - PVP Polyvinylpyrolidone     

Isolation and characterization of polymorphic microsatellites in the genome of Yak (Bos grunniens)

The researches on yak genetics and breeding were extremely restricted due to lacking of reliable DNA molecular markers. The microsatellites with repeat motif (AC)_n/(GT)_n in yak genome were enriched by Dynal magnetic beads and the gene libraries containing (AC)_n/(GT)_n were constructed. Among the 92 identified and sequenced positive clones, 40 contained perfect repeats (43.48 %), 41 contained imperfect repeats (44.57 %) and 11 contained compound repeats (11.96 %). As compared with the percentage of perfect repeats, no significant increases of imperfect repeats were observed in yak genome, which indicated that the level of adaptive evolution of the ability to repair damaged genomic DNA for yaks were high enough to endure the natural pressure of nucleotide substitution resulted from ultraviolet irradiation in high-altitude areas. Totally 19 polymorphic microsatellite loci were screened and genotyped on the basis of electropherograms on an ABI 310 Genetic Analyzer. All the loci exhibited moderate to high-level polymorphisms in a test population of Bos grunniens and the polymorphic information content ranged from 0.299 to 0.861 (mean 0.678). The newly isolated (AC)_n/(GT)_n repeats from yak genome will display their potential values in examining intra-population genetic structure and inter-population relationships, and also in investigating molecular markers for production and adaptive traits of individual/population.     

Isolation and characterization of 13 polymorphic microsatellites for the black murex, Hexaplex nigritus

Thirteen polymorphic microsatellite markers have been isolated and characterized for the black murex (Hexaplex nigritus). These loci are moderately to highly variable with seven to 37 alleles within 113 individuals from four populations in the Northern Gulf of California. Expected heterozygosities ranged from 0.43 to 0.98. High variability indicates that these markers will be useful for studying population structure and connectivity in this species.     

Isolation and characterization of polymorphic microsatellites for the bastard sole (Microchirus azevia)

The bastard sole (Microchirus azevia) is a species of commercial interest in Spain. Nevertheless, little information is currently available about the genetic characteristics of wild populations. In this survey, we have developed eight new microsatellites using an enriched genome library protocol. Primers were screened on a total of 54 individuals from two wild populations (Mediterranean and Atlantic) from the south coast of Spain, revealing six to 18 alleles per locus with expected heterozygosities ranging from 0.51 to 0.94. These markers can potentially be useful tools for use in population genetic studies.