Molecular phylogeny of mangroves V. Analysis of genome relationships in mangrove species using RAPD and RFLP markers

 DNA from pooled leaf samples of 11 true major mangrove, three true minor mangrove, two mangrove associate, two mangrove parasite, three terrestrial and one cultivated species were isolated for the present study. In total, 198 random amplified polymorphic DNAs (RAPDs) and 180 restriction fragment length polymorphism (RFLP) loci were scored by using ten primers and 14 enzyme-probe combinations respectively. The polymorphism observed for these markers revealed a high degree of genetic diversity in mangroves at both inter-specific or inter-generic levels. A dendrogram, constructed after pooling both RAPD and RFLP data, using a similarity index was analysed for genome relationships among these species. The dendrogram showed clustering of all the major mangroves, except for Nypa fruticans (Arecaceae), into one group. All species under the tribe Rhizophorae formed a sub-cluster, to which Xylocarpus granatum was found to be the most closesly related species. The clustering pattern implied that Excoecaria agallocha and Acanthus ilicifolius should be considered as true minor mangroves. The present study also provided molecular data favouring the separation of Avicennia spp. from the Verbenaceae to create a monotypic family the Avicenniaceae. The separation of Viscum orientale into the Viscaceae was also favoured. Received: 5 March 1998 / Accepted: 1 April 1998     

Molecular phylogeny of mangroves. VI. Intraspecific genetic variation in mangrove species Excoecaria agallocha L. (Euphorbiaceae).

Genomic DNA from 84 individuals of Excoecaria agallocha from seven mangrove populations were analysed for random amplified polymorphic DNAs (RAPDs) using 16 random 10-mer primers. Polymorphism within populations varied from 20% to 31%. At the interpopulation level, 111/149 (74%) of RAPDs were polymorphic. Restriction fragment length polymorphism (RFLP) analysis of 21 individuals (3 individuals randomly selected from the 7 populations) using 30 probe-enzyme combinations revealed a high level of interpopulation polymorphism (62.2%) indicating interpopulation genetic divergence. The polymorphic RAPDs and RFLPs were pooled, and clustering was carried out based on mean similarity for individual populations. The dendrogram showed groupings of populations from the West and East Coasts of India into separate clusters, at 60% similarity level. Further, RAPD and RFLP analysis of male and female plants showed approximately the same level of variation in both sexes, and no sex-linked markers were found. These results demonstrate that considerable intrapopulation and interpopulation genetic variations exist in E. agallocha, and that lack of genetic variation is not the reason for the morphological uniformity observed across the range of the species.     

Applicability of inter-simple sequence repeat polymorphisms in wheat for use as DNA markers in comparison to RFLP and RAPD markers

 Inter-simple sequence repeat polymorphic DNA (ISSR) was evaluated for its applicability as a genetic marker system in wheat. PCR was carried out with primers that annealed to simple sequence repeats. The resultant products were subjected to agarose-gel electrophoresis, and the banding patterns were compared among six wheat accessions containing diploid, tetraploid, and hexaploid members. Out of 100 examined, 33 primers produced distinguishable as well as polymorphic bands in each of the six accessions. Although most of the primers that gave distinct bands (30 primers out of 33) contained dinucleotide repeats, each of the primers with tri-, tetra-, and penta-nucleotide motifs also yielded discrete bands. Primers based on (AC)_n repeats gave the most polymorphic bands. In total, 224 polymorphic bands were found in the comparison between Einkorn wheats whereas, on the average, 120 polymorphic bands were detected between common wheats. ISSR primers produced several times more information than RAPD markers. The extent of band polymorphism was similar to that of RFLP markers, and greater than that of RAPDs. The genetic relationships of wheat accessions estimated by the polymorphism of ISSR markers were identical with those inferred by RFLP and RAPD markers, indicating the reliability of ISSR markers for estimation of genotypes. These polymorphic bands are potential candidates as novel markers for use in linkage-map construction in wheat. The characteristic features of ISSR markers, i.e. polymorphism, generation of information and ease of handling, suggest their applicability to the analysis of genotypes as well as to the construction of PCR-based genome maps of wheats. Received: 15 September 1996 / Accepted: 25 October 1996     

Genetic Relationship of <Emphasis Type="Italic">Curcuma</Emphasis> Species from Northeast India Using PCR-Based Markers

Molecular genetic fingerprints of nine Curcuma species from Northeast India were developed using PCR-based markers. The aim involves elucidating there intra- and inter-specific genetic diversity important for utilization, management, and conservation. Twelve random amplified polymorphic DNA (RAPD), 19 Inter simple sequence repeats (ISSRs), and four amplified fragment length polymorphism (AFLP) primers produced 266 polymorphic fragments. ISSR confirmed maximum polymorphism of 98.55% whereas RAPD and AFLP showed 93.22 and 97.27%, respectively. Marker index and polymorphic information content varied in the range of 8.64–48.1, 19.75–48.14, and 25–28 and 0.17–0.48, 0.19–0.48, and 0.25–0.29 for RAPD, ISSR, and AFLP markers, respectively. The average value of number of observed alleles, number of effective alleles, mean Nei’s gene diversity, and Shannon’s information index were 1.93–1.98, 1.37–1.62, 0.23–0.36, and 0.38–0.50, respectively, for three DNA markers used. Dendrograms based on three molecular data using unweighted pair group method with arithmetic mean (UPGMA) was congruent and classified the Curcuma species into two major clusters. Cophenetic correlation coefficient between dendrogram and original similarity matrix were significant for RAPD (r = 0.96), ISSR (r = 0.94), and AFLP (r = 0.97). Clustering was further supported by principle coordinate analysis. High genetic polymorphism documented is significant for conservation and further improvement of Curcuma species.     

RAPD markers for constructing intraspecific tomato genetic maps

The existing molecular genetic maps of the tomato, Lycopersicon spp, are constructed based on isozyme and RFLP polymorphisms between tomato species. These maps are useful for certain applications but have few markers that exhibit sufficient polymorphisms for intraspecific analysis and manipulations within the cultivated tomato. The purpose of this study was to investigate the relative potential of RAPD technology, as compared to isozymes and RFLPs, to generate polymorphic DNA markers within cultivated tomatoes. Sixteen isozymes and 25 RFLP clones that were known to detect polymorphism between L. esculentum and L. pennellii, and 313 random oligonucleotide primers were examined. None of the isozymes and only four of the RFLP clones (i.e., 16%) revealed polymorphism between the cultivated varieties whereas up to 63% of the RAPD primers detected one or more polymorphic DNA fragments between these varieties. All RAPD primers detected polymorphism between L. esculentum and L. pennellii genotypes. These results clearly indicate that RAPD technology can generate sufficient genetic markers exploiting sequence differences within cultivated tomatoes to facilitate construction of intraspecific genetic maps.Abbreviations RFLP restriction fragments length polymorphism - RAPD random amplified polymorphic DNA - PCR polymerase chain reaction - QTLs quantitative trait loci     

Evaluation of the extent of genetic variability among Theobroma cacao accessions using RAPD and RFLP markers

 Random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP) markers were used to evaluate genetic relationships within the Theobroma cacao species and to assess the organization of its genetic diversity. Genetic variability was estimated with 18 primers and 43 RFLP probes on 155 cocoa trees belonging to different morphological groups and coming from various geographic origins. The majority of the RFLP probes issued from low-copy DNA sequences. On the basis of on the genetic distance matrices, the two molecular methods gave related estimates of the genetic relationship between genotypes. Although an influence of cocoa morphological groups and geographical origins of trees was observed, a lack of gene differentiation characterized the T. cacao accessions studied. The continuous RFLP variability observed within the species may reflect the hybridization and introgressions between trees of different origins. Nevertheless, the Nacional type was detected to be genetically specific and different from well-known types such as Forastero, Criollo and Trinitario. Some of those genotypes were characterized by a low heterozygosity rate and may constitute the original Nacional pool. These results also provide information for the constitution of a cocoa tree core collection. Received: 10 June 1996/Accepted: 11 October 1996     

Comparative evaluation of genetic diversity using RAPD,SSR and cytochrome P450 gene based markers with respect to calcium content in finger millet (<Emphasis Type="Italic">Eleusine coracana</Emphasis> L. Gaertn.)

Genetic relationships among 52 Eleusine coracana (finger millet) genotypes collected from different districts of Uttarakhand were investigated by using randomly amplified polymorphic DNA (RAPD), simple sequence repeat (SSR) and cytochrome P450 gene based markers. A total of 18 RAPD primers, 10 SSR primers, and 10 pairs of cytochrome P450 gene based markers, respectively, revealed 49.4%, 50.2% and 58.7% polymorphism in 52 genotypes of E. coracana. Mean polymorphic information content (PIC) for each of these marker systems (0.351 for RAPD, 0.505 for SSR and 0.406 for cyt P450 gene based markers) suggested that all the marker systems were effective in determining polymorphisms. Pair-wise similarity index values ranged from 0.011 to 0.999 (RAPD), 0.010 to 0.999 (SSR) and 0.001 to 0.998 (cyt P450 gene based markers) and mean similarity index value of 0.505, 0.504 and 0.499, respectively. The dendrogram developed by RAPD, SSR and cytochrome P450 gene based primers analyses revealed that the genotypes are grouped in different clusters according to high calcium (300–450 mg/100 g), medium calcium (200–300 mg/100 g) and low calcium (100–200 mg/100 g). Mantel test employed for detection of goodness of fit established cophenetic correlation values above 0.95 for all the three marker systems. The dendrograms and principal coordinate analysis (PCA) plots derived from the binary data matrices of the three marker systems are highly concordant. High bootstrap values were obtained at major nodes of phenograms through WINBOOT software. Comparison of RAPD, SSR and cytochrome P450 gene based markers, in terms of the quality of data output, indicated that SSRs and cyt P450 gene based markers are particularly promising for the analysis of plant genome diversity. The genotypes of finger millet collected from different districts of Uttarakhand constitute a wide genetic base and clustered according to calcium contents. The identified genotypes could be used in breeding programmes and amajor input into conservation biology of cereal crops.     

Targeted identification of markers linked to malaria and filarioid nematode parasite resistance genes in the mosquito Aedes aegypti.

Quantitative trait loci (QTL) have been identified for competence of the mosquito Aedes aegypti to transmit the avian malaria parasite Plasmodium gallinaceum and the human filarial parasite Brugia malayi. Efforts towards the map-based cloning of the associated genes are limited by the availability of genetic markers for fine-scale mapping of the QTL positions. Two F2 mosquito populations were subjected to bulked segregant analysis to identify random amplified polymorphic DNA (RAPD)-PCR fragments linked with the major QTL determining susceptibility to both parasites. Individual mosquitoes for the bulks were selected on the basis of their genotypes at restriction fragment length polymorphism (RFLP) loci tightly linked with the QTL. Pool-positive RAPD fragments were cloned and evaluated as RFLP markers. Of the 62 RAPD/RFLP fragments examined, 10 represented low-copy number sequences. Five of these clones were linked with the major QTL for P. gallinaceum susceptibility (pgs1), of which one clone mapped within the flanking markers that define the QTL interval. The remaining five clones were linked with the major QTL for B. malayi susceptibility (fsb1), and again one clone mapped within the flanking markers that define the QTL interval. In addition, nine RAPD/RFLP fragments were isolated that seem to be of non-mosquito origin.     

RAPD Fingerprint to Appraise the Genetic Fidelity of In Vitro Propagated <Emphasis Type="Italic">Araucaria excelsa</Emphasis> R. Br. var. glauca Plantlets

Randomly amplified polymorphic DNA (RAPD) was used as a tool to assess the genetic fidelity of in vitro propagated Araucaria excelsa R. Br. var. glauca with explants taken from orthotropic stem along with their related mother plants after treatment with kinetin, 2iP, BA (0.02–0.26 mg/l) and TDZ (0.001–1 mg/l) to produce axillary shoots. TDZ and kinetin induced more shoot and higher length per explant. Results showed a total of 1,676 fragments were generated with 12 RAPD primers in micropropagated plants and their donor mother plants. The number of loci ranged from 6 in OPB 12–18 in OPY 07 with a size ranging from 250 bp in OPH 19–3500 bp in OPH 11. Cluster analysis of RAPD data using UPGMA (unweighted pair group method with arithmetic average) revealed more than 92% genetic similarities between tissue cultured plants and their corresponding mother plant measured by the Jaccard’s similarity coefficient. Similarity matrix and PCoA (two dimensional principal coordinate analysis) resulted in the same affinity. Primers had shown 36% polymorphism. However, careful monitoring of tissue culture derived plants might be needed to determine that rooted shoots are adventitious in origin.     

Genetic loci associated with field resistance to late blight in offspring of Solanum phureja and S.tuberosum grown under short-day conditions

Field resistance to late blight – a fungal disease caused by Phytophthora infestans – has been genetically characterized by analyzing trait-marker association in a Solanum phureja (phu)×dihaploid Solanum tuberosum (dih-tbr) population. Trait data were developed at three locations over a 3-year period under natural infection pressure. RAPD (random amplified polymorphic DNA) and AFLP (amplified fragment length polymorphism)markers were used to develop anonymous genetic linkage groups subsequently anchored to potato chromosomes using mapped RFLP (restriction fragment length polymorphism), SSR (single sequence repeats) and AFLP markers. RFLP and SSR markers achieved the most-accurate anchoring. Two genetic maps were obtained, with 987.4 cM for phu and 773.7 cM for dih-tbr. Trait-marker association was revealed by single-marker and interval mapping analyses. Two important QTLs (quantitative trait loci) were detected on chromosomes VII and XII as a contribution from both parents, totalling up to 16% and 43%, respectively, of the phenotypic variation (PH). One additional QTL was detected on chromosome XI (up to 11% of the PH) as a contribution from the phu parent, and three others were detected on chromosome III (up to 13% of the PH), chromosome V (up to 11% of the PH) and chromosome VIII (up to 11% of the PH) as a contribution from the dih-tbr parent. Our results reveal new genetic loci of the potato genome that contribute to resistance to late blight. We postulate that some of these loci could be related to plant growth under short-day conditions. Received: 5 July 2000 / Accepted: 17 November 2000     

SPAR methods revealed high genetic diversity within populations and high gene flow of Vanda coerulea Griff ex Lindl (Blue Vanda), an endangered orchid species

Molecular genetic fingerprints of seven populations of Vanda coerulea comprising of thirty-two genotypes from Northeast India were developed using PCR based markers. Genetic variability in the wild genotypes of V. coerulea was analyzed using two different single primer amplification reactions (SPAR) methods, viz., random amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR). A total of 32 genotypes were used to investigate the existing natural genetic diversity at intra-specific level. Two hundred and twenty six (226) amplification products were scored by RAPD and ISSR, both of which collectively showed 58.88% polymorphism with a mean intra-population genetic diversity (Hpop) of 0.119. However, their level of diversity at inter- and intra-population levels was significant, with the percentage of polymorphic loci (Pp) ranging from 17.70% to 45.13%, Shannon's information index (I) from 0.105 to 0.268 and Nei's gene diversity (h) from 0.072 to 0.185 with mean Nei's gene diversity 0.174 and the overall estimate of gene flow being (Nm) 1.165. Analysis of molecular variance (AMOVA) showed 96.07% of variation at intra-population level, whereas 3.93% variation was recorded at inter-population level. Only one major cluster was detected by cluster analysis using the unweighted pair-group method with arithmetic average (UPGMA). Present investigation suggests the efficiency of SPAR methods to estimate the genetic diversity of V. coerulea and can be seen as a starting point for future research on the population and evolutionary genetics of this species.     

Analysis of genetic diversity through AFLP,SAMPL, ISSR and RAPD markers in <Emphasis Type="Italic">Tribulus terrestris</Emphasis>, a medicinal herb

Tribulus terrestris is well known for its medicinal importance in curing urino-genital disorders. Amplified fragment length polymorphism (AFLP), selective amplification of microsatellite polymorphic loci (SAMPL), inter-simple sequence repeat (ISSR) and randomly amplified polymorphic DNA (RAPD) markers were used for the first time for the detection of genetic polymorphism in this medicinal herb from samples collected from various geographical regions of India. Six assays each of AFLP and SAMPL markers and 21 each of ISSR and RAPD markers were utilized. AFLP yielded 500 scorable amplified products, of which 82.9% were polymorphic. SAMPL primers amplified 488 bands, 462 being polymorphic (94.7%). The range of amplified bands was 66 [(TC)₈G + M-CAG] to 98 [(CA)₆AG + M-CAC] and the percentage polymorphism, 89.9 [from (CT)₄C (AC)₄A + M-CTG] to 100 [from (GACA)₄ + M-CTA]. The ISSR primers amplified 239 bands of 0.4–2.5 kb, 73.6% showed polymorphism. The amplified products ranged from 5 to 16 and the percentage polymorphism 40–100. RAPD assays produced 276 bands, of which 163 were polymorphic (59%). Mantel test employed for detection of goodness of fit established cophenetic correlation values above 0.9 for all the four marker systems. The dendrograms and PCA plots derived from the binary data matrices of the four marker systems are highly concordant. High bootstrap values were obtained at major nodes of phenograms through WINBOOT software. The relative efficiency of the four molecular marker systems calculated on the basis of multiplex ratio, marker index and average heterozygosity revealed SAMPL to be the best. Distinct DNA fingerprinting profile, unique to every geographical region could be obtained with all the four molecular marker systems. Clustering can be a good indicator for clear separation of genotypes from different regions in well-defined groups that are supported by high bootstrap values.     

A Comparative Analysis of ISSR and RAPD Markers for Study of Genetic Diversity in Shisham (<Emphasis Type="Italic">Dalbergia sissoo</Emphasis>)

Shisham (Dalbergia sissoo) is one of the most preferred timber tree species of South Asia. Two DNA-based molecular marker techniques, intersimple sequence repeat (ISSR) and random amplified polymorphism DNA (RAPD), were compared to study the genetic diversity in this species. A total of 30 polymorphic primers (15 ISSR and 15 random) were used. Amplification of genomic DNA of 22 genotypes, using ISSR analysis, yielded 117 fragments, of which 64 were polymorphic. Number of amplified fragments with ISSR primers ranged from five to ten and varied in size from 180 to 1,900 bp. Percentage polymorphism ranged from 0 to 87.5. The 15 RAPD primers produced 144 bands across 22 genotypes, of which 84 were polymorphic. The number of amplified bands varied from five to 13, with size range from 180 to 2,400 bp. Percentage polymorphism ranged from 0 to 100, with an average of 58.3 across. RAPD markers were relatively more efficient than the ISSR assay. The mental test between two Jaccard’s similarity matrices gave r ≥ 0.90, showing very good fit correlation in between ISSR- and RAPD-based similarities. Clustering of isolates remained more or less the same in RAPD and combined data of RAPD and ISSR. The similarity coefficient ranged from 0.734 to 0.939, 0.563 to 0.946, and 0.648 to 0.920 with ISSR, RAPD, and combined dendrogram, respectively.     

Evidence for new nuclear and mitochondrial genome organizations among high-frequency somatic embryogenesis-derived plants of allotetraploid Coffea arabica L. (Rubiaceae)

 The most important commercial species of coffee, Coffea arabica, which produces 73% of the world's coffee crop and almost all of the coffee in Latin America, is the only tetraploid (allotetraploid, 2n=4x=44) species known in the genus. High-frequency somatic embryogenesis, plant regeneration and plant recovery were achieved from leaf explants of a mature, elite plant of C. arabica cv. Cauvery (S-4347) using a two-step culture method. To assess the genetic integrity of the nuclear, mitochondrial and chloroplast genomes among the hardened regenerants, we employed multiple DNA markers (RFLP, RAPD, ISSR) for sampling various regions of the genome. Although the nuclear and mitochondrial genomes of the mother plant and five ramets derived from the mother ortet were similar in organization, this was not so in the somatic embryo-derived plants where both nuclear and mitochondrial genomes changed in different, characteristic ways and produced novel genome organizations. A total of 480 genetic loci, based on the data obtained from a total of 16 nuclear, mitochondrial and chloroplast gene probes, in combination with nine restriction enzyme digests, 38 RAPD and 17 SSR primers, were scored in 27 somatic embryo-derived plants and the single control. Among these, 44 loci were observed to be polymorphic. A relatively low level of polymorphism (4.36%) was found in the nuclear genome, while polymorphism in the mitochondrial genome (41%) was much higher. No polymorphism was detected in the chloroplast genome. The polymorphism in the mitochondrial genome was found in only 4 plants. Such selective polymorphism was not true for the nuclear genome. Thus, this in-depth and comprehensive study demonstrates, for the first time, the presence of subtle genetic variability and novel genome organizations in the commercially well-established somatic embryogenesis-derived plants of this important coffee species. Received: 2 July 1999 / Revision received: 1 February 2000 / Accepted: 17 February 2000     

Efficiency of RFLP, RAPD, and AFLP markers for the construction of an intraspecific map of the tomato genome.

We have constructed a tomato genetic linkage map based on an intraspecific cross between two inbred lines of Lycopersicon esculentum and L. esculentum var. cerasiforme. The segregating population was composed of 153 recombinant inbred lines. This map is comprised of one morphological, 132 RFLP (restriction fragment length polymorphism, including 16 known-function genes), 33 RAPD (random amplified polymorphic DNA), and 211 AFLP (amplified fragment length polymorphism) loci. We compared the 3 types of markers for their polymorphism, segregation, and distribution over the genome. RFLP, RAPD, and AFLP methods revealed 8.7%, 15.8%, and 14.5% informative bands, respectively. This corresponded to polymorphism in 30% of RFLP probes, 32% of RAPD primers, and 100% of AFLP primer combinations. Less deviation from the 1:1 expected ratio was obtained with RFLP than with AFLP loci (8% and 18%, respectively). RAPD and AFLP markers were not randomly distributed over the genome. Most of them (60% and 80%, respectively) were grouped in clusters located around putative centromeric regions. This intraspecific map spans 965 cM with an average distance of 8.3 cM between markers (of the framework map). It was compared to other published interspecific maps of tomato. Despite the intraspecific origin of this map, it did not show any increase in length when compared to the high-density interspecific map of tomato.     

Development and characterization of microsatellite markers for a mangrove tree species Sonneratia caseolaris (L.) Engler (Lythraceae sensu lato)

Sonneratia caseolaris is a typical non-viviparous mangrove species and a key component of mangrove community in the Indo-West Pacific region. Here we isolated nine microsatellite simple sequence repeat (SSR) loci from the genome of S. caseolaris. Our isolated loci provided SSR markers with polymorphism of 2–6 alleles per locus. The expected and observed heterozygosities ranged from 0.242 to 0.745 and from 0.083 to 0.417, respectively. Cross-species amplification in S. alba and S. ovata showed that a subset of these markers holds promise for these congeneric species. These polymorphic SSR markers would be useful tools for population genetics studies on S. caseolaris as well as other congeneric species.     

Linkage map of Japanese black pine based on AFLP and RAPD markers including markers linked to resistance against the pine needle gall midge

Macrogametophytes derived from the seeds of a tree resistant to pine needle gall midge (PGM) were analyzed using amplified fragment length polymorphism (AFLP). A total of 244 segregating loci were detected among 71 macrogametophytes. Combining the AFLP results with previously reported segregation data for 127 random amplified polymorphic DNA (RAPD) markers, 157 AFLP and 50 RAPD markers with confirmed map positions were assigned to 20 linkage groups and three pairs covering 2085.5 cM with an average distance of 10.1 cM. The total map distance covers about 77.1–78.4% of the total genome, estimated to be approximately 2665–2719 cM in length. Thus, using AFLP markers, the previous RAPD map of this tree was improved in terms of the average distance between markers, the total map distance, and coverage of the genome. Three RAPD markers linked to a gene associated with resistance to PGM were also located on this map. Rceived: 14 April 2000 / Accepted: 21 August 2000     

DNA polymorphisms in grain sorghum (Sorghum bicolor (L.) Moench)

Molecular markers [random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP)] were used to determine the frequency of DNA polymorphism in grain sorghum (Sorghum bicolor (L.) Moench). Twenty-nine oligonucleotide primers were employed for RAPDs, generating a total of 262 DNA fragments, of which 145 were polymorphic in at least one pairwise comparison between 36 genotypes. Individual primers differed significantly in their ability to detect genetic polymorphism in the species. The overall frequency of polymorphisms was low with a mean frequency of 0.117 polymorphisms per RAPD band being obtained from all pairwise comparisons between genotypes, with maximum and minimum values of 0.212 and 0.039, respectively. Results from phenetic analysis of bandsharing data were consistent with current sub-specific groupings of the species, with clusters of Durra, Zerazera, Caud-Nig, Caud-Kaura and Caffrorum being discernible. The results also indicated that individuals of a similar taxonomic grouping but different geographic origin may be genetically less identical than previously considered. Similar frequencies of polymorphism to that obtained with RAPDs were obtained with RFLPs. Results from these experiments indicated that a high level of genetic uniformity exists within S. bicolor.     

Development of microsatellite markers for the white mangrove (Laguncularia racemosa C.F. Gaertn., Combretaceae)

We developed nine new microsatellite markers for the white mangrove (Laguncularia racemosa C.F. Gaertn.), a mangrove species widely distributed in the Western Hemisphere. The loci were developed from a sample collected in Baja California Sur, Mexico, and were screened for polymorphism in 108 individuals from the Pacific Coast of Mexico and Costa Rica. The level of polymorphism was relatively low, from two to four alleles. These are the first set of polymorphic markers described for L. racemosa and should be of value in studying regional genetic differentiation among natural populations of this species.     

Evaluation of inter-simple sequence repeat analysis for mapping in Citrus and extension of the genetic linkage map

Inter-simple sequence repeat (ISSR) analysis was evaluated for its usefulness in generating markers to extend the genetic linkage map of Citrus using a backcross population previously mapped with restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD) and isozyme markers. ISSR markers were obtained through the simple technique of PCR followed by analysis on agarose gels, using simple sequence repeat (SSR) primers. Optimization of reaction conditions was achieved for 50% of the SSR primers screened, and the primers amplified reproducible polymorphic bands in the parents and progeny of the backcross population. Mendelian segregation of the polymorphic bands was demonstrated, with an insignificant number of skewed loci. Most of the SSR primers produced dominant loci; however co-dominance was observed with loci derived from three primers. A new genetic map was produced by combining the segregation data for the ISSR markers and data for the RFLP, RAPD and isozyme markers from the previous map and creating genetic linkages among all the markers using JoinMap 2.0 mapping software. The new map has an improved distribution of markers along the linkage groups with fewer gaps, and marker order showed partial or complete conservation in the linkage groups. The incorporation of ISSR markers into the genetic linkage map demonstrates that ISSR markers are suitable for genetic mapping in Citrus. Received: 3 February 2000 / Accepted: 12 May 2000