Suitability of AFLP markers for the study of genetic relationships among Korean native dogs.

To determine the genetic relationships among domestic dog breeds, we performed both a sequence comparison of mitochondrial DNA (mtDNA) and an amplified fragment length polymorphisms (AFLP) analysis. Three of four regions of mtDNA, cytochrome b, cytochrome oxidase subunit II, and 16S rRNA genes were highly homogeneous among dog breeds, whereas the other region, the control region, showed relatively high polymorphisms with a maximum percentage difference of 3.18%. However, the control region showed extensive polymorphism even within breeds, and the relationship tree derived from the data could not clearly delimit distinct breeds. 19 EcoRI/MseI primer combinations were used to generate AFLP markers among 25 dogs from 11 breeds including three Korean native dogs. These amplification reactions allowed the detection of more than 1900 amplification products of which 408 were identified as polymorphic bands. Unrooted neighbor-joining tree based on dissimilarity values showed that the Korean native dogs were clustered together with the Asian dogs and that the Asian originated dogs were clustered separately from Western originated dogs. A consensus tree using parsimony method also showed Korean native dogs were grouped separately from the other dogs with moderate bootstrap values. Taken together, it is concluded that AFLP analysis is a more informative tool for revealing genetic relationships among dog breeds than mtDNA sequence comparison.     

Identification of mitochondrial markers for genetic traceability of European wild boars and Iberian and Duroc pigs

Iberian pigs and wild boars are the source of highly priced meat and dry-cured products. Iberian maternal origin is mandatory for labeled Iberian products, making necessary the authentication of their maternal breed origin. Discrimination between wild and domestic pig maternal origin may be useful to distinguish labeled wild boar meat obtained from hunting or farming. In order to detect useful polymorphisms to trace Iberian, Duroc and wild boar maternal lineages, we herein investigated the complete porcine mitochondrial DNA (mtDNA) using three complementary approaches. Near-complete mtDNA sequences (16989 bp), excluding the minisatellite present in the displacement loop region (D-loop), were successfully determined in six Iberian pigs, two Duroc and six European wild boars. To complete the mtDNA analysis, the D-loop minisatellite region was also analyzed in the same set of samples by amplification and capillary electrophoresis detection. Finally, the frequencies of Asian and European Cytochrome B (Cyt B) haplotypes were estimated in Iberian (n = 96) and Duroc (n = 125) breeds. Comparison of near-complete mtDNA sequences revealed a total of 57 substitutions and two Indels. Out of them, 32 polymorphisms were potential Iberian markers, 10 potential Duroc markers and 16 potential wild boar markers. Fourteen potential markers (five Iberian and nine Duroc), were selected to be genotyped in 96 Iberian and 91 Duroc samples. Five wild boar potential markers were selected and tested in samples of wild boars (73) and domestic pigs including: 96 Iberian, 16 Duroc, 16 Large White and 16 Landrace. Genotyping results showed three linked markers (m.7998C>T, m.9111T>C, m.14719A>G) absent in Duroc and present in Iberian pigs with a frequency 0.72. Six markers (m.8158C>T, m.8297T>C, m.9230G>A, m.11859A>G, m.13955T>C, m.16933T>C), three of them linked, were absent in Iberian pigs and present in Duroc with a joint frequency of almost 0.50. Finally three linked markers (m.7188G>A, m.9224T>C, m.15823A>G) were solely detected in wild boars with a frequency 0.22. The D-loop minisatellite results showed overlapping ranges of fragment sizes and suggested heteroplasmy, a result that nullify the use of this region for the development of breed diagnostic markers. The Cyt B haplotype results showed the presence of European haplotypes in Iberian while one of the Asian haplotypes was detected in Duroc with a frequency 0.22, linked to the Duroc marker m.9230G>A. Our results are valuable to resolve the problems of Iberian and wild boar maternal origin determination but additional markers are required to achieve totally useful genetic tests.     

Intraspecific Differentiation of Vibrio vulnificus Biotypes by Amplified Fragment Length Polymorphism and Ribotyping

The intraspecific genomic relatedness of 80 Vibrio vulnificus isolates, 44 of biotype 1 and 36 of biotype 2, from different geographic origins and sources was evaluated by ribotyping and AFLP (amplified fragment length polymorphism) fingerprinting. Ribopatterns of DNAs digested with KpnI and hybridized with an oligonucleotide complementary to a highly conserved sequence in the 23S rRNA gene revealed up to 19 ribotypes in the species, which were different for the two biotypes. Sixteen different ribotypes were found within biotype 1 strains from clinical and environmental sources, and only three, recovered mainly from diseased eels, were found within biotype 2. Within this biotype, 96% of the strains showed the same ribopattern. The closest similarity was shown by the strains coming from the same eel farm, irrespectively of biotype. AFLP fingerprints obtained by selective PCR amplification of HindIII-TaqI double-restricted DNA fragments exhibited a strain-specific pattern which allowed the finest differentiation of subgroups within the eel-pathogenic isolates sharing the same ribopattern. Both techniques revealed good genetic markers for intraspecific differentiation of V. vulnificus. Ribotyping clearly separated the eel-pathogenic strains from the clinical and environmental isolates, whereas AFLP enabled the monitoring of individual strains and therefore constitutes one of the most discriminative tools for epidemiological and ecological studies.     

The inheritance and chromosomal localization of AFLP markers in a non-inbred potato offspring

AFLP^TM is a new technique to generate large numbers of molecular markers for genetic mapping. The method involves the selective amplification of a limited number of DNA restriction fragments out of complex plant genomic DNA digests using PCR. With six primer combinations 264 segregating AFLP amplification products were identified in a diploid backcross population from non-inbred potato parents. The identity of an AFLP marker was specified by the primer combination of the amplification product and its size estimated in bases. The segregating AFLP amplification products were mapped by using a mapping population with 217 already known RFLP, isozyme and morphological trait loci. In general, the AFLP markers were randomly distributed over the genome, although a few clusters were observed. No indications were found that AFLP markers are present in other parts of the genome than those already covered by RFLP markers. Locus specificity of AFLP markers was demonstrated because equally sized amplification products segregating from both parental clones generally mapped to indistinguishable maternal and paternal map positions. Locus specificity of AFLP amplification products will allow to establish the chromosomal identity of linkage groups in future mapping studies.Since AFLP technology is a multi-locus detection system, it was not possible to identify the AFLP alleles which belong to a single AFLP locus. The consequences of a genetic analysis based on single alleles, rather than on loci with two or more alleles on mapping studies using progenies of non-inbred parents are discussed.     

Molecular structure in peripheral dog breeds: Portuguese native breeds as a case study

Genetic variability in purebred dogs is known to be highly structured, with differences among breeds accounting for ∼30% of the genetic variation. However, analysis of the genetic structure in non-cosmopolitan breeds and local populations is still limited. Nine Portuguese native dog breeds, and other peripheral dog populations (five) with regional affinities, were characterized using 16 microsatellites and 225 amplified fragment length polymorphism (AFLP) markers, and the pattern of genetic differentiation was investigated. Although the level of breed differentiation detected is below that of other dog breeds, there is in most cases a correlation between breed affiliation and molecular structure. AFLP markers and Bayesian clustering methods allowed an average of 73.1% of individuals to be correctly assigned to source populations, providing robust genotypic assessment of breed affiliation. A geographical genetic structure was also detected, which suggests a limited influence of African dogs on the Iberian breeds. The sampling effect on the estimation of population structure was evaluated and there was a 2.2% decrease in genetic differentiation among breeds when working animals were included. Genetic diversity of stray dogs was also assessed and there is no evidence that they pose a threat to the preservation of the gene pool of native dog breeds.     

Strain-specific differentiation of lactococci in mixed starter culture populations using randomly amplified polymorphic DNA-derived probes.

A hydrophobic grid membrane filtration (HGMF) colony hybridization assay was developed that allows strain-specific differentiation of defined bacterial populations. The randomly amplified polymorphic DNA (RAPD) fingerprinting technique was used to identify potential signature nucleic acid sequences unique to each member of a commercial cheese starter culture blend. The blend consisted of two closely related Lactococcus lactis subsp. cremoris strains, 160 and 331, and one L. lactis subsp. lactis strain, 210. Three RAPD primers (OPX 1, OPX 12, and OPX 15) generated a total of 32 products from these isolates, 20 of which were potential strain-specific markers. Southern hybridization analyses revealed, that the RAPD-generated signature sequences OPX15-0.95 and a 0.36-kb HaeIII fragment of OPX1-1.0b were specific for strains 331 and 210, respectively, within the context of the test starter culture blend. These strain-specific probes were used in a HGMF colony hybridization assay. Colony lysis, hybridization, and nonradioactive detection parameters were optimized to allow specific differentiation and quantitation of the target strains in the mixed starter culture population. When the 210 and 331 probes were tested at their optimal hybridization temperatures against single cultures, they detected 100% of the target strain CFUs, without cross-reactivity to the other strains. The probes for strains 210 and 331 also successfully detected their targets in blended cultures even with a high background of the other two strains.     

Chemiluminescent detection of AFLP markers

Nonradioactive amplified fragment-length polymorphism (AFLP) marker detection, a PCR-based, DNA-fingerprinting technique, was achieved by blotting AFLP products after electrophoresis onto a nylon membrane and subsequently hybridizing the blot with an alkaline phosphatase-labeled AFLP probe. Similar AFLP profiles were obtained by both a nonradioactive, chemiluminescent detection technique and by conventional AFLP marker detection using 32P-labeled AFLP primers. The suitability of the method using different gel systems combined with subsequent chemiluminescent detection of AFLP markers is validated by similar dendrograms that were generated using the unweighted pair group method with arithmetic averages (UPGMA). Moreover, chemiluminescent detection of AFLP markers using a universal AFLP nonradioactive probe has been successfully applied on prokaryotes such as Agrobacterium and eukaryotic genomes such as soybean and fungi.     

A potato hypersensitive resistance gene against potato virus X maps to a resistance gene cluster on chromosome 5

 The dominant Nb gene of potato confers strain-specific hypersensitive resistance against potato virus X (PVX). A population segregating for Nb was screened for resistance by inoculating with PVX strain CP2, which is sensitive to Nb. Through a combination of bulked segregant analysis and selective restriction fragment amplification, several amplified fragment length polymorphism (AFLP) markers linked to Nb were identified. These were cloned and converted into dominant cleaved amplified polymorphic sequence (CAPS) markers. The segregation of these markers in a Lycopersicon esculentum×L. pennellii mapping population suggested that Nb is located on chromosome 5. This was confirmed by examining resistant and susceptible potato individuals with several tomato and potato chromosome-5-specific markers. Nb maps to a region of chromosome 5 where several other resistance genes– including R1, a resistance gene against Phytophthora infestans, Gpa, a locus that confers resistance against Globodera pallida, and Rx2, a gene that confers extreme resistance against PVX–have previously been identified. Received: 2 January 1997/Accepted: 7 February 1997     

Estimating levels of inbreeding using AFLP markers

In the absence of detailed pedigree records, researchers have attempted to estimate individuals' levels of inbreeding using molecular markers, generally making use of heterozygosity measures based on microsatellite markers. Here we report and validate a method for estimating an individual's inbreeding coefficient, f, using amplified fragment length polymorphism (AFLP) markers. We use simulations to confirm that our measure scales appropriately with f when allele frequencies can be estimated from a subset of outbred individuals. We also present an approach for obtaining satisfactory estimates even in the absence of an independent set of known outbred individuals from which to estimate allele frequencies. We then test our method against empirical data from 179 wild and captive-bred old-field mice, Peromyscus polionotus subgriseus, comprising pedigree-based estimates of f, along with genetic data from 94 AFLP markers and 12 microsatellites. Inbreeding estimates based on both AFLP and microsatellite markers were found to correlate strongly with pedigree-based inbreeding coefficients. Owing to their ease of amplification in any species, AFLP markers may prove to be a valuable new tool for estimating f in natural populations and for examining correlations between heterozygosity and fitness.     

Genetic characterization of Erwinia amylovora strains by amplified fragment length polymorphism

AIMS: Erwinia amylovora is one of the most important pathogens of pear and apple and is subject to strict quarantine regulations worldwide, although its patterns of dispersal are largely unknown. Previous attempts to fingerprint E. amylovora strains by molecular techniques have detected very little polymorphism because of the high genetic homogeneity of this bacterium. Our aim was to establish and test a typing method to quantify genetic diversity among strains of this plant pathogen. METHODS AND RESULTS: Twenty-two strains from different hosts and geographical locations were examined by PCR fingerprinting with four primers and by amplified fragment length polymorphism (AFLP) with four selected combinations of primers with a single base extension. PCR fingerprinting revealed little polymorphism producing the same amplification patterns for 17 strains, while the combined AFLP patterns yielded 78 polymorphic bands (34% of total bands) and allowed the differentiation of all but two strains. Clustering of strains in the resulting dendrogram was not correlated with host, year or country of isolation, and questions previous genealogies based on PFGE patterns. CONCLUSIONS: The AFLP technique allowed the detection of an unprecedented number of genetic markers in E. amylovora and proved to be the most useful tool so far for discriminating among strains of this pathogen. The results obtained in this study strongly suggest the occurrence of multiple introductions of the pathogen in Spain and other European countries. SIGNIFICANCE AND IMPACT OF THE STUDY: A major limitation in understanding the ecology of fire blight is the lack of typing techniques with a high power of discrimination. This study demonstrates the high resolution and the usefulness of the AFLP technique to differentiate among E. amylovora strains.     

Female‐only sex‐linked amplified fragment length polymorphism markers support ZW/ZZ sex determination in the giant freshwater prawn Macrobrachium rosenbergii

Sex determination mechanisms in many crustacean species are complex and poorly documented. In the giant freshwater prawn, Macrobrachium rosenbergii, a ZW/ZZ sex determination system was previously proposed based on sex ratio data obtained by crosses of sex‐reversed females (neomales). To provide molecular evidence for the proposed system, novel sex‐linked molecular markers were isolated in this species. Amplified fragment length polymorphism (AFLP) using 64 primer combinations was employed to screen prawn genomes for DNA markers linked with sex loci. Approximately 8400 legible fragments were produced, 13 of which were uniquely identified in female prawns with no indication of corresponding male‐specific markers. These AFLP fragments were reamplified, cloned and sequenced, producing two reliable female‐specific sequence characterized amplified region (SCAR) markers. Additional individuals from two unrelated geographic populations were used to verify these findings, confirming female‐specific amplification of single bands. Detection of internal polymorphic sites was conducted by designing new primer pairs based on these internal fragments. The internal SCAR fragments also displayed specificity in females, indicating high levels of variation between female and male specimens. The distinctive feature of female‐linked SCAR markers can be applied for rapid detection of prawn gender. These sex‐specific SCAR markers and sex‐associated AFLP candidates unique to female specimens support a sex determination system consistent with female heterogamety (ZW) and male homogamety (ZZ).     

Development of AFLP markers in barley

To investigate the application of amplified fragment length polymorphism (AFLP) markers in barley, 96 primer combinations were used to generate AFLP patterns with two barley lines, L94 and Vada. With seven primer combinations, only a few intense bands were obtained, probably derived from repeated sequences. With the majority of the remaining 89 primer combinations, on average about 120 amplification products were generated, and the polymorphism rate between the two lines was generally over 18%. Based on the number of amplified products and the polymorphism rate, the 48 best primer combinations were selected and tested on 16 barley lines, again including L94 and Vada. Using a subset of 24 primer combinations 2188 clearly visible bands within the range from 80 to 510 bp were generated; 55% of these showed some degree of polymorphism among the 16 lines. L94 versus Vada showed the highest polymorphism rate (29%) and Proctor versus Nudinka yielded the lowest (12%). The polymorphism rates per primer combination showed little dependence on the barley lines used. Hence the most efficient and informative primer combinations identified for a given pair of lines turned out to be highly efficient when applied to others. Generally, more than 100 common markers (possibly locus specific) among populations or crosses were easily identified by comparing 48 AFLP profiles of the parent lines. The existence of such a large number of markers common to populations will facilitate the merging of molecular marker data and other genetic data into one integrated genetic map of barley. Received: 28 October 1996 / Accepted: 27 November 1996     

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.     

Molecular identification of AFLP fragments associated with elytra color variation of the Asian ladybird beetle,Harmonia axyridis (Coleoptera: Coccinellidae)

The Asian ladybird beetle, Harmonia axyridis shows polymorphism in elytra color patterns. However, it is uncertain whether these color patterns are regulated by genetic factors. This investigation used amplified fragment length polymorphism (AFLP) analysis to determine any genetic causes of the variability of color patterns. Using four individuals of each group, AFLP analysis produced 37 polymorphic bands. Among several polymorphic bands, six AFLP markers were associated with elytra color patterns after further analysis using six additional individuals of each group. These polymorphic sites were sequenced but did not match DNA sequence data deposited in GenBank. Based on the color-associated AFLP markers, SCAR primers were designed for PCR amplification of genomic DNA. These primers (SCAR 12 and SCAR 44) were used to analyze color-associated loci and/or alleles of H. axyridis DNA. SCAR 12 primers designed from a Spectabilis type-specific fragment (AFLP 12) amplified a specific band of 530 bp in four Spectabilis individuals, but not in the insects with other color patterns.     

Genetic divergence of Trichogramma aurosum Sugonjaev and Sorokina (Hymenoptera: Trichogrammatidae) individuals based on ITS2 and AFLP analysis

Taxonomy and phylogeny of members of the genus Trichogramma is often critical because of the fact that proper species discrimination can only be achieved by male morphology. Cryptic species, particularly when only females are available (in case of parthenogenetic species or strains), are common in this genus with consequences for practical purposes like biocontrol, unless males can be obtained after antibiotic treatment. The internally transcribed spacer 2 region of the ribosomal DNA was used to assess the identity of Trichogramma aurosum Sugonjaev and Sorokina individuals collected on eggs of Nematus tibialis Newman (Hymenoptera: Tenthredinidae) from different locations in Middle Europe. Amplified products were identical in length (ca. 450bp), sequences showed a high percent similarity (>96%), and no cryptic species could be detected in the samples. In contrast, a comparison with T. aurosum populations from the USA showed values between 86% and 90%. Additional studies are needed to clarify the relationship between US and European populations. Furthermore, amplified fragment length polymorphism (AFLP) analysis was conducted with T. aurosum wasps collected at 25 different European locations. One hundred and twenty‐three AFLP fragments could be detected using three different AFLP primer combinations of which 98% were polymorphic in more than one individual. An analysis of genetic distances based on the obtained AFLP markers indicated the existence of some genetic variability between the European T. aurosum individuals and allowed a grouping according to their geographic origin. This study represents the first successful application of the AFLP marker technique to such tiny insects as Trichogramma species.     

Development of AFLP-derived, functionally specific markers for environmental persistence studies of fungal strains

The ability to rapidly identify and quantify a microbial strain in a complex environmental sample has widespread applications in ecology, epidemiology, and industry. In this study, we describe a rapid method to obtain functionally specific genetic markers that can be used in conjunction with standard or real-time polymerase chain reaction (PCR) to determine the abundance of target fungal strains in selected environmental samples. The method involves sequencing of randomly cloned AFLP (amplified fragment length polymorphism) products from the target organism and the design of PCR primers internal to the AFLP fragments. The strain-specific markers were used to determine the fate of three industrially relevant fungi, Aspergillus niger, Aspergillus oryzae, and Chaetomium globosum, during a 4 month soil microcosm experiment. The persistence of each of the three fungal strains inoculated separately into intact soil microcosms was determined by PCR analyses of DNA directly extracted from soil. Presence and absence data based on standard PCR and quantification of the target DNA by real-time PCR showed that all three strains declined after inoculation (approximately 14-, 32-, and 4-fold for A. niger, A. oryzae, and C. globosum, respectively) but remained detectable at the end of the experiment, suggesting that these strains would survive for extended periods if released into nature.     

PCR-based molecular markers for assessment of somaclonal variation in <Emphasis Type="Italic">Pinus pinea</Emphasis> clones micropropagated <Emphasis Type="Italic">in vitro</Emphasis>

Four different markers [random amplified polymorphic DNA (RAPD), inter simple sequence repeat (ISSR), amplified fragment length polymorphism (AFLP), and selective amplified microsatellite polymorphism length (SAMPL)] were applied for evaluating somaclonal variation of micropropagated genotypes of stone pine (Pinus pinea L.). The total number of primers tested was 130, with 223 combinations assayed. A high number of them amplified successfully (178), representing 79.82 % of the total, and the average number of amplified fragments ranged from 2.47 (ISSR) to 65.76 (SAMPL). Based on internal controls, no problem of reproducibility was detected. Almost no somaclonal variation was detected within the clones. Of the tested markers, ISSR, AFLP, and SAMPL showed monomorphic amplification profiles, with only RAPD markers showing some interclonal variation.     

Conversion of AFLP markers to sequence-specific markers for closely related lines in rice by use of the rice genome sequence

DNA polymorphism between two major japonica rice cultivars, Nipponbare and Koshihikari, was identified by AFLP. Eighty-four polymorphic AFLP markers were obtained by analysis with 360 combinations of primer pairs. Nucleotide sequences of 73 markers, 29 from Nipponbare and 44 from Koshihikari, were determined, and 46 AFLP markers could be assigned to rice chromosomes based on sequence homology to the rice genome sequence. Specific primers were designed for amplification of the regions covering the AFLP markers and the flanking sequences. Out of the 46 primer pairs, 44 amplified single DNA fragments, six of which showed different sizes between Nipponbare and Koshihikari, yielding codominant SCAR markers. Eight primer pairs amplified only Nipponbare sequences, providing dominant SCAR markers. DNA fragments amplified by 13 primer pairs showed polymorphism by CAPS, and polymorphism of those amplified by 13 other primer pairs were detected by PCR-RF-SSCP (PRS). Nucleotide sequences of the other four DNA fragments were determined in Koshihikari, but no difference was found between Koshihikari and Nipponbare. In total, 40 sequence-specific markers for the combination of Nipponbare and Koshihikari were produced. All the SNPs identified by AFLP were detectable by CAPS and PRS. The same method was applicable to a combination of Kokoromachi and Tohoku 168, and 23 polymorphic markers were identified using these two rice cultivars. The procedure of conversion of AFLP-markers to the sequence-specific markers used in this study enables efficient sequence-specific marker production for closely related cultivars.     

Mitochondrial DNA control region polymorphisms: genetic markers for ecological studies of marine turtles

We describe a rapid and sensitive method for the detection of population-specific genetic markers in mitochondrial DNA (mtDNA) and the use of such markers to analyse population structure of marine turtles. A series of oligonucleotide primers specific for the amplification of the mtDNA control region in Cheloniid turtles were designed from preliminary sequence data. Using two of these primers, a 384–385-bp sequence was amplified from the 5′ portion of the mtDNA control region of 15 green turtles Chelonia mydas from 12 different Indo-Pacific rookeries. Fourteen of the 15 individuals, including some with identical whole-genome restriction fragment patterns, had sequences that differed by one or more base substitutions. Analysis of sequence variation among individuals identified a total of 41 nucleotide substitutions and a 1-bp insertion/deletion. Comparison with evidence from whole-genome restriction enzyme analysis of the same individuals indicated that this portion of the control region is evolving approximately eight times faster than the average rate and that the sequence analysis detected approximately one fifth of the total variation present in the genome. Restriction enzyme analysis of amplified products from an additional 256 individuals revealed significant geographic structuring in the distribution of mtDNA genotypes among five of the 10 rookeries surveyed extensively. Additional geographic structuring of genotypes was identified through denaturing gradient gel electrophoresis (DGGE) of amplified products. Only two of the 10 rookeries surveyed could not be differentiated, indicating that the Indo-Pacific C. mydas include a number of genetically differentiated populations, with minimal female-mediated gene flow among them. Important applications for genetic markers in the conservation and management of marine turtles include the identification of appropriate demographic units for research and management (i.e. genetically discrete populations) and assessment of the composition of feeding and harvested populations.     

Identification ofporphyra lines (rhodophyta) by AFLP DNA fingerprinting and molecular markers

Twenty-sevenPorphyra lines from 5 classes, including lines widely used in China, wild lines, and lines introduced to China from abroad in recent years, were screened by means of amplified fragment length polymorphism (AFLP) with 24 primer pairs. From the generated AFLP products, 13 bands that showed stable and repeatable AFLP patterns amplified by primer pairs M-CGA/E-AA and M-CGA/E-TA were scored and used to develop the DNA fingerprints of the 27Porphyra lines. Moreover, the DNA fingerprinting patterns were converted into computer language expressed with digitals 1 and 0, which represented the presence (numbered as 1) or absence (numbered as 0) of the corresponding band. On the basis of these results, computerized AFLP DNA fingerprints were constructed in which each of the 27Porphyra lines has its unique AFLP fingerprinting pattern and can be easily distinguished from others. Software called PGI-AFLP (Porphyra germ-plasm identification-AFLP) was designed for identification of the 27Porphyra lines. In addition, 21 specific AFLP markers from 15Porphyra lines were identified; 6 AFLP markers from 4Porphyra lines were sequenced, and 2 of them were successfully converted into SCAR (sequence characterized amplification region) markers. The developed AFLP DNA fingerprinting and specific molecular markers provide useful ways for the identification, classification, and resource protection of thePorphyra lines.