Identification, genetic localization, and allelic diversity of selectively amplified microsatellite polymorphic loci in lettuce and wild relatives (Lactuca spp.).

Selectively amplified microsatellite polymorphic locus (SAMPL) analysis is a method of amplifying microsatellite loci using generic PCR primers. SAMPL analysis uses one AFLP primer in combination with a primer complementary to microsatellite sequences. SAMPL primers based on compound microsatellite sequences provided the clearest amplification patterns. We explored the potential of SAMPL analysis in lettuce to detect PCR-based codominant microsatellite markers. Fifty-eight SAMPLs were identified and placed on the genetic map. Seventeen were codominant. SAMPLs were dispersed with RFLP markers on 11 of the 12 main linkage groups in lettuce, indicating that they have a similar genomic distribution. Some but not all fragments amplified by SAMPL analysis were confirmed to contain microsatellite sequences by Southern hybridization. Forty-five cultivars of lettuce and five wild species of Lactuca were analyzed to determine the allelic diversity for codominant SAMPLs. From 3 to 11 putative alleles were found for each SAMPL; 2-6 alleles were found within Lactuca sativa and 1-3 alleles were found among the crisphead genotypes, the most genetically homogeneous plant type of L. sativa. This allelic diversity is greater than that found for RFLP markers. Numerous new alleles were observed in the wild species; however, there were frequent null alleles. Therefore, SAMPL analysis is more applicable to intraspecific than to interspecific comparisons. A phenetic analysis based on SAMPLs resulted in a dendrogram similar to those based on RFLP and AFLP markers.     

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.     

利用AFLP和SSAP研究短芒大麦突变系种群遗传多样性

利用AFLP和SSAP分子标记,研究了松嫩平原4个以耐盐突变体为主要组成的短芒大麦(Hordeum brevisubulatium(Trin.)Link)种群的遗传多样性和种群遗传结构。由于2种分子标记原理稍有不同,获得分析结果显现出微小差异,但2种方法的结果仍保持了极显著的相关性(r=0.88,P<0.01)。AFLP和SSAP检测结果表明,短芒大麦种群多态位点比率P分别为23.3%和37.7%;Shannon多样性指数分别为0.10和0.14;Nei基因多样性指数分别为0.07和0.09;基因分化系数Gst分别为0.49和0.44。表明,各短芒大麦种群虽以单一突变种为主要组成,但经多年的种植,种群内已具有较高的遗传多样性。AMOVA和聚类分析结果也表明,虽然种群间遗传差异较大,但种群遗传变异仍主要存在于种群内。     

Relationships among fourteen species of Satureja growing wild in Iran detected with molecular markers

The genus Satureja is an important plant with a number of aromatic and medicinal properties. In this research, the relative efficiencies of amplified fragment length polymorphism (AFLP) and selectively amplified microsatellite polymorphic loci (SAMPL) were used to detect genetic relationships among 14 species of Satureja, growing wild in Iran. Eleven AFLP and 14 SAMPL primer combinations produced 999 and 1142 scorable bands, respectively, all of the fragments of which were found to be polymorphic. The average genetic similarity values based on Jaccard's coefficient were 0.24 and 0.21 for AFLP and SAMPL, respectively, indicating considerable distance and diversity in the studied germplasm. The correlation coefficients were statistically significant between both marker systems (r = 0.89). UPGMA derived from the combined binary data matrices of both markers depicted genetic distinctions among the studied species and clustered them into two main clusters and several groups. S. edmondi showed the maximum distance from other species and was placed into a single main cluster, while the maximum similarity was obtained between S. rechingeri and S. khuzistanica. Our results indicate that both marker systems are suitable for differentiating individuals and species of this genus.     

Efficiency of three PCR-based markers in assessing genetic variation among cowpea (Vigna unguiculata subsp. unguiculata) landraces.

The main objective of this study was to investigate the efficiency of RAPD, AFLP, and SAMPL marker systems in detecting genetic polymorphism in cowpea landraces (Vigna unguiculata subsp. unguiculata (L.) Walp.) that probably share a similar genetic pool. A second objective was to determine the level of diversity among landraces from a restricted area, to define the most appropriate strategy of on-farm conservation. Each marker system was able to discriminate among the materials analysed, but a clear distinction between all the local varieties was only obtained with AFLP and SAMPL markers. The average diversity index was quite similar for each marker system, but owing to the differences in the effective multiplex ratio values the marker index was higher for the AFLP and SAMPL systems than for the RAPD system. The AFLP and SAMPL techniques appear to be more useful than the RAPD technique in the analysis of limited genetic diversity among the cowpea landraces tested. The significant correlations of SAMPL similarity and cophenetic matrices with those of the other markers, and the lower number of primer combinations required, indicate that this technique is the most valuable. The low genetic similarity detected among landraces suggests that all the cowpea landraces should be maintained on the respective farms from which they came.     

利用AFLP分子标记探讨蜡梅种质资源的遗传多样性

利用AFLP分子标记技术,对中国蜡梅种质资源7个野生种群的遗传多样性进行了研究。利用筛选出的3对引物,共扩增出253条谱带,其中218条多态带,多态位点占86.17% ;种群间的基因分化系数为0.2906,说明蜡梅基因多样性主要存在于种群内;种群总的Nei s基因多样性指数为0.2933,Shannon信息多态性指数为0.4487,蜡梅总的遗传多样性水平较高。蜡梅不同种群遗传多样性水平差异较大,种群多态位点百分率在65.44% ～87.16%之间,Nei s基因多样性指数为0.1653 ～0.4012,Shannon信息多态性指数为0.3132 ～0.5603。神农架种群(SN)和保康种群(BK)的遗传多样性水平较高。用NTSYS2.01版软件对样品进行UPGMA聚类分析,结果7个种群并没有按地理距离进行聚类。最后提出要对各蜡梅野生群体采取相应的迁地和就地保护措施。     

Intraspecific diversity and relationship between subspecies of Origanum vulgare revealed by comparative AFLP and SAMPL marker analysis

The genus Origanum is often referred to as an under-utilized taxon because of its complex taxonomy. Origanum vulgare L., the most variable species of the genus, is a spice and medicinal herb that is characterized by high morphological diversity (six subspecies). In this study, the relative efficiencies of two PCR-based marker approaches, amplified fragment length polymorphism (AFLP) and selectively amplified microsatellite polymorphic loci (SAMPL), were used for comparable genetic diversity surveys and subspecies discrimination among 42 oregano accessions. Seven assays each of AFLP and SAMPL markers were utilized. Effective multiplex ratio (EMR), average heterozygosity (H_av-p), marker index (MI), and resolving power (RP) of the primer combinations were calculated for the two marker systems. UPGMA and Structure analysis along with PCoA plots derived from the binary data matrices of the two markers depicted the genetic distinction of accessions. Our results indicate that both marker systems are suitable but SAMPL markers are slightly more efficient in differentiating accessions and subspecies than AFLPs.     

Development of S-SAP markers based on an LTR-like sequence from Medicago sativa L.

The Sequence-Specific Amplification Polymorphism (S-SAP) method, recently derived from the Amplified Fragment Length Polymorphism (AFLP) technique, produces amplified fragments containing a retrotransposon LTR sequence at one end and a host restriction site at the other. We report the application of this procedure to the LTR of the Tms1 element from Medicago sativa L. Genomic dot-blot analysis indicated that Tms1 LTRs represent about 0.056% of the M. sativa genome, corresponding to 16 x 10(3) copies per haploid genome. An average of 66 markers were amplified for each primer combination. Overall 49 polymorphic fragments were reliably scored and mapped in a F(1) population obtained by crossing diploid M. falcata with M. coerulea. The utility of the LTR S-SAP markers was higher than that of AFLP or SAMPL (Selective Amplification of Microsatellite Polymorphic Loci) markers. The efficiency index of the LTR S-SAP assay was 28.3, whereas the corresponding values for AFLP and SAMPL markers were 21.1 and 16.7, respectively. The marker index for S-SAP was 13.1, compared to 8.8 for AFLP and 9.5 for SAMPL. Application of the Tms1 LTR-based S-SAP to double-stranded cDNA resulted in a complex banding pattern, demonstrating the presence of Tms1 LTRs within exons. As the technique was successfully applied to other species of the genus Medicago, it should prove suitable for studying genetic diversity within, and relatedness between, alfalfa species.     

Genetic variation and population structure of oriental migratory locust, Locusta migratoria manilensis, in China by allozyme, SSRP-PCR, and AFLP markers

Allozyme analysis, microsatellite primer PCR (SSRP-PCR), and amplified fragment length polymorphism (AFLP) techniques were used to assess genetic diversity and population structure of the Chinese oriental migratory locust, Locusta migratoria manilensis. A total of 299 PCR markers (67 SSRPs and 232 AFLPs) were detected in eight populations, of which 98.7% were polymorphic markers. The proportion of polymorphic loci (95.5-98.8%) by SSRP+AFLP markers indicated no significant differences between populations, and all populations exhibited a similar level of variability; results of the allozyme analysis demonstrated that 19 loci gave rise to a lower level of polymorphism (55.6-66.7%). The genetic distances between the populations were relatively low. Shannon's index and Nei's gene diversity showed low differentiation among the populations. Allozyme analysis, however, reflected greater similarity and smaller differentiation between the populations than those shown by SSRP and AFLP markers. Neighbor-joining dendrograms derived from both the allozyme and SSRP+AFLP markers showed that the genetic distances among Chinese oriental migratory locust populations were not greatly influenced by geographic distance and breeding habitats.     

Genetic Variation and Population Structure of Oriental Migratory Locust, Locusta migratoria manilensis, in China by Allozyme, SSRP-PCR, and AFLP Markers

Allozyme analysis, microsatellite primer PCR (SSRP-PCR), and amplified fragment length polymorphism (AFLP) techniques were used to assess genetic diversity and population structure of the Chinese oriental migratory locust, Locusta migratoria manilensis. A total of 299 PCR markers (67 SSRPs and 232 AFLPs) were detected in eight populations, of which 98.7% were polymorphic markers. The proportion of polymorphic loci (95.5–98.8%) by SSRP+AFLP markers indicated no significant differences between populations, and all populations exhibited a similar level of variability; results of the allozyme analysis demonstrated that 19 loci gave rise to a lower level of polymorphism (55.6–66.7%). The genetic distances between the populations were relatively low. Shannon’s index and Nei’s gene diversity showed low differentiation among the populations. Allozyme analysis, however, reflected greater similarity and smaller differentiation between the populations than those shown by SSRP and AFLP markers. Neighbor-joining dendrograms derived from both the allozyme and SSRP+AFLP markers showed that the genetic distances among Chinese oriental migratory locust populations were not greatly influenced by geographic distance and breeding habitats.     

Genomic organization of molecular differentiation in Norway spruce (Picea abies)

Diversity and differentiation among three populations representing the geographical domains commonly recognized within the natural distribution area of Picea abies were analysed by using a set of 292 AFLP (amplified fragment length polymorphism), SSR (single sequence repeat) and ESTP (expressed sequence tags polymorphism) markers. As usually observed in forest trees, results showed high within-population diversity (H(S) reaching 0.79) and low among-population differentiation (G(ST) approximately 2%). The genomic organization of differentiation was then investigated on the basis of a subsample of 150 AFLP, SSR and ESTP mapped markers. The number of the loci differentiating the Baltico-Nordic from the central European populations (25 loci) and, within the central European populations, the Alpine from the Hercyno-Carpathian populations (12 loci), were different. These 37 differentiated loci, with individual G(ST) values ranging from 0.008 to 0.20, were evenly distributed on all linkage groups and mostly followed the neutral expectations, suggesting genome-wide effects on differentiation. Nine of them however behave as 'outlier' loci indicating possible locus-specific selective effects. Contribution of ongoing evolutionary forces and historical effects to the geographical differentiation of the species are discussed.     

Genetic diversity in an endangered alpine plant, Eryngium alpinum L. (Apiaceae), inferred from amplified fragment length polymorphism markers

Eryngium alpinum L. is an endangered species found across the European Alps. In order to obtain base-line data for the conservation of this species, we investigated levels of genetic diversity within and among 14 populations from the French Alps. We used the amplified fragment length polymorphism (AFLP) technique with three primer pairs and scored a total of 62 unambiguous, polymorphic markers in 327 individuals. Because AFLP markers are dominant, within-population genetic structure (e.g. FIS) could not be assessed. Analyses based either on the assumption of random-mating or on complete selfing lead to very similar results. Diversity levels within populations were relatively high (mean Nei's expected heterozygosity = 0.198; mean Shannon index = 0.283), and a positive correlation was detected between both genetic diversity measurements and population size (Spearman rank correlation: P = 0. 005 and P = 0.002, respectively). Moreover, FST values and exact tests of differentiation revealed high differentiation among populations (mean pairwise FST = 0.40), which appeared to be independent of geographical distance (nonsignificant Mantel test). Founder events during postglacial colonizations and/or bottlenecks are proposed to explain this high but random genetic differentiation. By contrast, we detected a pattern of isolation by distance within populations and valleys. Predominant local gene flow by pollen or seed is probably responsible for this pattern. Concerning the management of E. alpinum, the high genetic differentiation leads us to recommend the conservation of a maximum number of populations. This study demonstrates that AFLP markers enable a quick and reliable assessment of intraspecific genetic variability in conservation genetics.     

AFLP and SAMPL markers for characterization of genetic diversity in Terminalia arjuna: a backbone tree of Tasar silk industry

Overexploitation of Terminalia arjuna by the Tasar silk, pharmaceutical, leather, and other industries has led to gradual depletion of its germplasm resource and genetic diversity. Information on its existing gene pool is currently lacking. We report the first evaluation of genetic variation in this tree, using amplified fragment length polymorphism (AFLP) and selective amplification of microsatellite polymorphic loci (SAMPL) markers. The study consisted of (1) characterization of genetic diversity at interzonal and intrazonal levels, (2) comparison of diversity within and among populations, and (3) comparison of efficiency of the two marker systems. The germplasm used in the present study had a wide genetic base, which is evident through the high level of genetic diversity observed, as expected for a widespread, long-lived tropical species. The clustering obtained after interzonal genetic diversity analysis of T.?arjuna is not region specific, a reflection of the lack of any defined population structure in this genus in India. The high value of r obtained through Mantel??s test for evaluation of cophenetic correlation in cluster analysis indicated the high fitness of the accessions to a group. The region-specific bands obtained during the present investigation can be used as diagnostic markers for authentication and to identify raw materials for herbal drug preparations.     

Genetic diversity among Japanese indigenous common buckwheat (Fagopyrum esculentum) cultivars as determined from amplified fragment length polymorphism and simple sequence repeat markers and quantitative agronomic traits.

We assessed the genetic diversity in Japanese indigenous common buckwheat (Fagopyrum esculentum) cultivars using amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers and investigated the relationships between the genetic diversity and agronomic traits. The average expected intracultivar hetero zygosity was 0.303 for AFLP and 0.819 for SSR. The differentiations among agroecotypes, among cultivars within an agroecotype, and among cultivars were small (0.002, 0.024, and 0.026 for SSR and 0.013, 0.013, and 0.026 for AFLP, respectively) but statistically significant from zero except for the SSR differentiation among agroecotypes. In principal coordinates analysis, cultivars within the same agroecotype tended to cluster, indicating that agroecotypes well reflected the genetic relationships among cultivars. In AFLP, the differentiation among the agroecotypes was more distinct than in SSR, and genetic distance showed a moderate correlation with the difference in quantitative traits, indicating that AFLP can resolve the relationships among cultivars with better resolution than SSR. By contrast, SSR may be more sensitive to demographic changes. Four of the five SSR markers showed a significant positive correlation (Kendall's tau = 0.382-0.607) between allelic richness and variation in flowering timing, indicating that cumulative bottleneck events have occurred during the population history, with a decline in the variation of photosensitivity of flowering.     

AFLP and RAPD analyses of genetic diversity of wild and/or weedy Guizotia (Asteraceae) from Ethiopia

Amplified fragment length polymorphism (AFLP) and random amplified polymorphic DNA (RAPD) markers were used to provide estimates of the comparative genetic variation within and among populations of various Guizotia taxa with the goal of conserving and utilizing their genetic diversity. The percentage of polymorphic loci (P(S)) ranged from 28.5%-90% (AFLP) and 85.6%-99.6% (RAPD). The overall gene diversity estimate () has shown slight variation among taxa ranging from 0.32-0.37 (AFLP) and from 0.22 to 0.28 (RAPD). The within population diversity of "Chelelu" and "Ketcha" was found to be unexpectedly high. Both parameters used to estimate population differentiation (G(ST) and F(ST)) revealed the highest population differentiation G. zavattarii in followed by G. arborescens. Genetic variation among populations within a taxon was highly significant for all the five taxa as revealed by AMOVA (P<0.0001). The need for immediate conservation activities for G. arborescens and G. zavattarii, and factors that contribute to the existing genetic variability and population genetic structures are discussed.     

A population genetics study of Anopheles darlingi (Diptera: Culicidae) from Colombia based on random amplified polymorphic DNA-polymerase chain reaction and amplified fragment lenght polymorphism markers

The genetic variation and population structure of three populations of Anopheles darlingi from Colombia were studied using random amplified polymorphic markers (RAPDs) and amplified fragment length polymorphism markers (AFLPs). Six RAPD primers produced 46 polymorphic fragments, while two AFLP primer combinations produced 197 polymorphic fragments from 71 DNA samples. Both of the evaluated genetic markers showed the presence of gene flow, suggesting that Colombian An. darlingi populations are in panmixia. Average genetic diversity, estimated from observed heterozygosity, was 0.374 (RAPD) and 0.309 (AFLP). RAPD and AFLP markers showed little evidence of geographic separation between eastern and western populations; however, the F ST values showed high gene flow between the two western populations (RAPD: F ST = 0.029; Nm: 8.5; AFLP: F ST = 0.051; Nm: 4.7). According to molecular variance analysis (AMOVA), the genetic distance between populations was significant (RAPD:phiST = 0.084; AFLP:phiST = 0.229, P < 0.001). The F ST distances and AMOVAs using AFLP loci support the differentiation of the Guyana biogeographic province population from those of the Chocó-Magdalena. In this last region, Chocó and Córdoba populations showed the highest genetic flow.     

Random amplified polymorphic DNA and amplified fragment length polymorphism assessment of genetic variation in Nicaraguan populations of Pinus oocarpa

Pinus oocarpa is the most widely distributed pine species of Mexico and Central America. The natural populations of Nicaragua have been affected by extensive human activities. As a consequence, their size has been reduced, and there is a serious threat to the development of mature woodland. Knowledge of population structures and the genetic diversity of the species is required for the design of sustainable use and conservation strategies. Random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) markers were used to assess the genetic variation among 10 populations from three geographical regions of Nicaragua. Both markers revealed high levels of diversity in these populations. G(ST) values and analyses of molecular variance (AMOVA) found that most variation was within populations but there is still a significant differentiation between populations indicating that the populations sampled cannot be considered a single panmictic unit. The partitions created by AMOVA also showed that there was little differentiation between populations of different regions, although cluster analyses based on RAPDs and AFLPs indicated a closer relationship among most of the populations from a same geographical region. Management of P. oocarpa in Nicaragua should be aimed to maintain the high degree of genetic variation within individual populations that is still observed even in some of these highly degraded populations.     

Comparison of 23S polymerase chain reaction-restriction fragment length polymorphism and amplified fragment length polymorphism techniques as typing systems for thermophilic campylobacters

In this study, we evaluated the combination of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and amplified fragment length polymorphism (AFLP) molecular typing techniques for the analysis of thermophilic campylobacter species isolated from clinical and poultry samples. 23S PCR-RFLP analysis performed to fingerprint 69 strains exhibited an excellent level of typability. Eleven different types were defined at 100% linkage level following numerical analysis of band patterns. Differentiation of Campylobacter jejuni and Campylobacter coli at species level was achieved although no significant relationship could be observed between the profiles and the origin of the strains. Simplified AFLP analysis of the isolates disclosed the presence of 66 different banding patterns. The resulting dendrogram showed a high diversity among the strains studied. All the isolates were grouped within eight main types with a 69% homology degree among them. Differentiation at subspecies level was possible but no significant relationship could be observed between the AFLP profiles and the origin of the strains. When used in combination, 23S PCR-RFLP and single-enzyme AFLP methods can be applied to determine taxonomic and epidemiological relationships among thermophilic campylobacters.     

Genetic differentiation analysis of African cassava (Manihot esculenta) landraces and elite germplasm using amplified fragment length polymorphism and simple sequence repeat markers

Molecular‐marker‐aided evaluation of germplasm plays an important role in defining the genetic diversity of plant genotypes for genetic and population improvement studies. A collection of African cassava landraces and elite cultivars was analysed for genetic diversity using 20 amplified fragment length polymorphic (AFLP) DNA primer combinations and 50 simple sequence repeat (SSR) markers. Within‐population diversity estimates obtained with both markers were correlated, showing little variation in their fixation index. The amount of within‐population variation was higher for landraces as illustrated by both markers, allowing discrimination among accessions along their geographical origins, with some overlap indicating the pattern of germplasm movement between countries. Elite cultivars were grouped in most cases in agreement with their pedigree and showed a narrow genetic variation. Both SSR and AFLP markers showed some similarity in results for the landraces, although SSR provided better genetic differentiation estimates. Genetic differentiation (Fst) in the landrace population was 0.746 for SSR and 0.656 for AFLP. The molecular variance among cultivars in both populations accounted for up to 83% of the overall variation, while 17% was found within populations. Gene diversity (H_e) estimated within each population varied with an average value of 0.607 for the landraces and 0.594 for the elite lines. Analyses of SSR data using ordination techniques identified additional cluster groups not detected by AFLP and also captured maximum variation within and between both populations. Our results indicate the importance of SSR and AFLP as efficient markers for the analysis of genetic diversity and population structure in cassava. Genetic differentiation analysis of the evaluated populations provides high prospects for identifying diverse parental combinations for the development of segregating populations for genetic studies and the introgression of desirable genes from diverse sources into the existing genetic base.     

Low genetic differentiation among seasonal cohorts in Senecio vulgaris as revealed by amplified fragment length polymorphism analysis

Common groundsel, Senecio vulgaris (Asteraceae), is a highly selfing semelparous ephemeral weed that belongs to the few plant species in central Europe capable of growing, flowering and fruiting all year round. In temperate climates, flowering S. vulgaris cohorts were found to appear up to three times per year. Using amplified fragment length polymorphism (AFLP) molecular markers we examined temporal genetic differentiation among spring, summer and autumn cohorts at each of seven sites located in two regions in Switzerland. Strong genetic differentiation among cohorts may indicate the existence of seasonal races of S. vulgaris, reproductively isolated by nonoverlapping flowering phenologies. Analysis of molecular variance (amova) revealed that < 2.5% of the AFLP variation resided among cohorts within sites, whereas there was significant genetic differentiation among plants from different sites (15.6%) and among individuals within cohorts (81.9%). Significant genetic differentiation was also observed between the two regions. Isolation-by-distance was found on a regional scale, but not on a local scale. Gene flow was estimated to be approximately 15-fold higher among cohorts within sites than among sites. We further found, on average, similar levels of genetic diversity within the three seasonal cohorts. The results of this study demonstrate that season of growth represents a weak barrier for genetic exchange among S. vulgaris populations and does not affect molecular variance. Therefore, there is no evidence for the existence of seasonally specialized races of S. vulgaris. We discuss some implications of the results for the biological control of S. vulgaris using a native rust fungus.