Comparison of RAPD and RFLP genetic markers in determining genetic similarity among Brassica oleracea L. genotypes

Genetic similarity among 45 Brassica Oleracea genotypes was compared using two molecular markers, random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphisms (RFLPs). The genotypes included 37 broccolis (var. italica), five cauliflowers (var. botrytis) and three cabbages (var. capitata) which represented a wide range of commercially-available germplasm, and included open-pollinated cultivars, commercial hybrids, and inbred parents of hybrid cultivars. Fifty-six polymorphic RFLP bands and 181 polymorphic RAPD bands were generated using 15 random cDNA probes and 62 10-mer primers, respectively. The objectives were to compare RFLP and RAPD markers with regard to their (1) sampling variance, (2) rank correlations of genetic distance among sub-samples, and (3) inheritance. A bootstrap procedure was used to generate 200 random samples of size n (n=2,3,5,... 55) independently from the RAPD and RFLP data sets. The coefficient of variance (CV) was estimated for each sample. Pooled regressions of the coefficient of variance on bootstrap sample size indicated that the rate of decrease in CV with increasing sample size was the same for RFLPs and RAPDs. The rank correlation between the Nei-Li genetic similarity values for all pairs of genotypes (990) based on RFLP and RAPD data was 0.745. Differences were observed between the RFLP and RAPD dendrograms of the 45 genotypes. Overlap in the distributions of rank correlations between independent sub-samples from the RAPD data set, compared to correlations between RFLP and RAPD sub-samples, suggest that observed differences in estimation of genetic similarity between RAPDs and RFLPs is largely due to sampling error rather than due to DNA-based differences in how RAPDs and RFLPs reveal polymorphisms. A crossing algorithm was used to generate hypothetical banding patterns of hybrids based on the genotypes of the parents. The results of this study indicate that RAPDs provide a level of resolution equivalent to RFLPs for detemination of the genetic relationships among genotypes.     

Genetic similarity among Arabidopsis thaliana ecotypes estimated by DNA sequence comparison

DNA polymorphisms among Arabidopsis thaliana ecotypes are widely used as genetic markers in map-based cloning strategies. New PCR-based molecular markers do not only facilitate molecular mapping, but can also be used to obtain reliable sequence information for cladistic analyses. We have used CAPS (cleaved amplified polymorphic sequences) markers and a direct sequencing strategy to estimate genetic similarity among eighteen Arabidopsis ecotypes. Sequences at four loci, two from the nuclear and two from a non-nuclaar genome, were analysed. For each ecotype more than 1000pb of sequence information was obtained, and genetic similarity was calculated from a total of 35 polymorphic sites using a character-based approach. Divergence ranged from zero up to 50 discordant characters among the 72 characters defined by the polymorphisms. Separate calculations based on the nuclear and the non-nuclear sequences were performed and revealed a number of common features, including the existence of small clusters of very closely related ecotypes separated from each other by extensive sequence divergence. Our results provide information useful especially to investigators setting up crosses for chromosome landing strategies.     

Estimating genetic diversity of Arabidopsis thaliana ecotypes with amplified fragment length polymorphisms (AFLP)

The extensive natural variation of Arabidopsis thaliana ecotypes is being increasingly exploited as a source of variants of genes which control (agronomically) important traits. We have subjected 19 different Arabidopsis thaliana ecotypes to an analysis using the anplified fragment length polymorphism (AFLP) technique in order to estimate their genetic diversity. The genetic diversity was estimated applying the method of Nei and Li (1979) and a modified version of it and using 471 informative polymorphisms. The data obtained revealed that within this small set of ecotypes a group of three ecotypes and a further single ecotype exhibit considerable genetic diversity in comparison to the others. These ecotypes clustered at positions significantly separated from the bulk of the ecotypes in the generated similarity plots. The analysis demonstrated the usefulness of the AFLP method for determinating intraspecies genetic diversity as exemplified with Arabidopsis thaliana ecotypes. Results are discussed and compared with data obtained with other methods. Received: 18 June 1999 / Accepted: 28 July 1999     

RFLP analyses of early-maturing European maize germ plasm

Summary Thirty inbred lines representing a wide range of early-maturing European elite germ plasm of maize (Zea mays L.) were assayed for RFLPs using 203 clone-enzyme combinations (106 DNA clones with restriction enzymes EcoR1 and HindIII). The genetic materials comprised 14 flint, 12 dent, and 4 lines of miscellaneous origin. Objectives were to (1) characterize the genetic diversity for RFLPs in these materials, (2) compare the level of genetic diversity found within and between the flint and the dent heterotic groups, and (3) examine the usefulness of RFLPs for assigning inbreds to heterotic groups. All but two DNA clones yielded polymorphism with at least one restriction enzyme. A total of 82 and 121 clone-enzyme combinations gave single-banded and multiple-banded RFLP patterns, respectively, with an average of 3.9 and 7.7 RFLP patterns per clone-enzyme combination across all 30 inbreds, respectively. Genetic similarity (GS) between lines, estimated from RFLP data as Dice's similarity coefficient, showed considerable variation (0.32 to 0.58) among unrelated inbreds. The mean GS for line combinations of type flint x dent (0.41) was significantly smaller than for unrelated flint lines (0.46) and dent lines (0.46), but there was considerable variation in GS estimates of individual line combinations within each group. Cluster and principal coordinate analyses based on GS values resulted in separate groupings of flint and dent lines in accordance with phylogenetic information. Positioning of lines of miscellaneous origin was generally consistent with expectations based on known breeding behavior and pedigrees. Results from this study corroborated that RFLP data can be used for assigning inbreds to heterotic groups and revealing pedigree relationships among inbreds.     

Estimation of sampling variance of molecular marker data using the bootstrap procedure

Knowledge of genetic relationships among genotypes is useful in a plant breeding program because it permits the organization of germplasm and provides for more efficient sampling. The genetic distance (GD) among genotypes can be estimated using random restriction fragment length polymorphisms (RFLPs) as molecular markers. Knowledge of the sampling variance associated with RFLP markers is needed to determine how many markers are required for a given level of precision in the estimate of GD. The sampling variance for GD among all pairs of 37 maize (Z. mays L.) inbred lines was estimated from 1202 RFLPs. The 1202 polymorphisms were generated from 251 enzyme-probe combinations (EPC). The sampling variance was used to determine how large a sample of RFLPs was required to provide a given level of precision. The coefficient of variation (CV) associated with GD has a nearly linear relationship between its expected standard deviation and mean. The magnitude of the decrease in the mean CV for GD with increasing numbers of bands was dependent upon the sampling unit; e.g., individual polymorphic bands vs EPC, and the degree of relatedness among the inbreds compared. The rate of reduction in mean CV with increasing sample size was the same regardless of the restriction enzyme used, BamHI, EcoRI or HindIII, when the bootstrap sampling units were individual polymorphic bands. In constrast, although the rate of reduction (slopes) was the same, the intercepts of the mean CVs were different when EPCs were used as the bootstrap sampling unit. This difference was due to the higher number of bands per EPC in BamHI (4.94) compared with EcoRI (4.83) and HindIII (4.63).     

Suitability of RAPD for analyzing spined and spineless variant regenerants of pineapple (Ananas comosus L., Merr.)

A random amplified polymorphic DNA (RAPD) analysis of spineless (variant phenotype) plants obtained from micropropagated dormant pineapple (Ananas comosus L., Merr.) axillary buds was performed using arbitrary 10-mer oligonucleotide primers. This was done to investigate the genetic fidelity of the regenerants and to distinguish these variants from regenerants bearing the normal spined phenotype. Of the 58 arbitrary primers used, 29 produced bands unique to the spineless phenotype, and 30 produced bands unique to the spined phenotype. A total of 914 bands were scored, 55 of which were polymorphic to the spineless phenotype and 51 of which were polymorphic to the spined phenotype. On the basis of RAPD amplification products, genetic similarity was estimated in both types of regenerants using similarity coefficients (Nei and Li, 1979). The characteristic finger-prints generated by each probe emphasize genetic variability of regenerants. This technique is suitable for analyzing variant regenerants induced in vitro.     

Estimation of genetic variation in Cynodon dactylon accessions using the ISSR technique

Genetic variation in 55 accessions of Cynodon dactylon was estimated using inter-simple sequence repeat (ISSR) markers. The plant materials used in this study originated from 17 countries. A total of 236 ISSR fragments were generated with 14 primers. Fragment sizes ranged from 200 to 3000 bp. All scorable bands were polymorphic in nature and none of the primers used produced monomorphic bands, indicating a high level of genetic variation in this grass. The accessions were found to be clustered into eight major groups through the unweighted pair-group method with arithmetic averages. Genetic similarity coefficients (GSC) among the 55 accessions ranged from 0.52 to 0.95. The results clearly indicate that a high level of variation exists in Cynodon accessions. This study shows that the ISSR technique is a reliable tool for differentiating Cynodon accessions and for determining the genetic relationships among them.     

Genetic diversity in a germplasm collection of roseroot (Rhodiola rosea) in Norway studied by AFLP

Rhodiola rosea is widely distributed in Norway, but so far limited knowledge exists on the level of genetic diversity. To initiate a selective breeding program, Amplified Fragment Length Polymorphism (AFLP) analysis was used to estimate genetic diversity within the Norwegian R. rosea germplasm collection. AFLP analysis of 97 R. rosea clones using five primer combinations gave a total of 109 polymorphic bands. We detected high percentage of polymorphic bands (PPB) with a mean of 82.3% among the clones of R. rosea. Each of the 97 R. rosea clones could be unambiguously identified based on these primer combinations. Estimates of genetic similarities were obtained by the Dice coefficient, and a final dendrogram was constructed with the Unweighted Pair Group Method with Arithmetic mean (UPGMA). Genetic similarity based on the AFLP data ranged from 0.440 to 0.950 with a mean of 0.631. This genetic analysis showed that there was no close genetic similarity among clones related to their original growing county. No gender-specific markers were found in the R. rosea clones. Analysis of molecular variance (AMOVA) revealed a significantly greater variation within regions (92.03%) than among regions (7.97%). A low level of genetic differentiation (F_ST = 0.043) was observed, indicating a high level of gene flow, which had a strong influence on the genetic structure at different counties. Our results indicate high gene flow among R. rosea clones that might be a result of seed dispersal rather than cross-pollination. Further world-wide studies are required to compare the level of genetic diversity and more studies in R. rosea detailing the consequences of different patterns of gene flow (pollen spread and dispersal of seeds and clonal plants) will be useful for characterization of roseroot.     

Genetic Diversity of Persian Ecotypes of Indian Walnut (<Emphasis Type="Italic">Aeluropus littoralis</Emphasis> (Gouan) Pari.) by AFLP and ISSR Markers

Indian walnut Aeluropus littoralis (Gouan) Pari, well known for its salt tolerance and forage quality, is a perennial monocot grass from Chlorideae tribe that shows a high potential to become an important genetic model. Although, Aeluropus germplasm is very rich in Iran; but is still unexplored. In this study, 20 ecotypes of A. littoralis were gathered from different geographic zones and a set 16 primers (ISSRs-AFLPs combined) was used for genetic diversity evaluation. A total of 635 fragments and 594 polymorphic fragments were produced (93.5%) and modest levels of genetic similarity were found (ranging from 0.38 to 0.71) among ecotypes. The results showed an equal competition between the two markers on polymorphism detection, so both molecular techniques were able to distinguish the A. littoralis ecotypes. Furthermore, Dendrogram based on both the Bayesian and PCoA analysis showed a clear discrimination and significant variation among ecotypes; however, molecular clustering indicated a weak concordance with geographical grouping. The amount of molecular diversity revealed by molecular markers for A. littoralis in this study can be exploited for pasture conservation programs and new forage crop development.     

Genetic diversity and relationships within sweet kernel apricot and related Armeniaca species based on sequence-related amplified polymorphism markers

Sequence-related amplified polymorphism (SRAP) markers were employed to detect the genetic variation among 28 sweet kernel apricot and related Armeniaca accessions from China. Fifteen SRAP primer combinations were used and 252 polymorphic bands out of 286 with an average of 16.8 per primer combination were observed. The unweighted pair-group method (UPGMA) analysis demonstrated that the accessions had a similarity range from 0.55 to 0.91 with a mean of 0.7, and there were three clusters with two groups of sweet kernel apricots distinctly separated from the related Armeniaca vulgaris and Armeniaca sibirica at the similarity level of 0.58, indicating more extensive genetic variation among sweet kernel apricot. The origin of sweet kernel apricot has also been discussed. This suggested there are abundant germplasm resources of sweet kernel apricot from China for further breeding and biodiversity conservation.     

Construction of Fingerprinting and Genetic Diversity of Mulberry Cultivars in China by ISSR Markers

The ISSR fingerprintings of 24 mulberry cultivars were constructed. Totally 80 bands were produced using 17 primers selected from 20 primers. Of them, 40 bands showed polymorphism. From the bands amplified, there were three independent ways to identify the mulberry varieties, such as unique ISSR markers, unique band patterns and a combination of the band patterns provided by different primers. ISSRs were very effective in differentiating the mulberry varieties. The mean genetic similarity coefficient, the mean Nei's gene diversity (h), and the mean Shannon's Information index (I) of mulberry cultivars were 0.8731, 0.1210, and 0.1942, respectively. This suggests that the genetic diversity of mulberry cultivars was low and the genetic base was narrow. Both UPGMA cluster and PCA (Principal Coordinates Analysis) analysis showed clear genetic relationships among the 24 mulberry cultivars. The major clusters were related to known pedigree relationships.     

Phylogenetic relationships between Leymus and related diploid Triticeae species revealed by ISSR markers

To investigate the genetic diversity and phylogenetic relationships between polyploid Leymus and related diploid species of the Triticeae tribe, inter-simple sequence repeats (ISSR) markers was used to analyze 41 Leymus accessions representing 22 species and 2 subspecies, together with Pseudoroegneria stipifolia (St), Psathyrostachys fragilis (Ns), Australopyrum retrofractum (W), Hordeum bogdanii, H. chilense (H) and Lophopyrum elongatum (E^e). A total of 376 clear and reproducible DNA fragments were amplified by 29 ISSR primers, among which 368 (97.87%) fragments were found to be polymorphic. 8–18 polymorphic bands were amplified by each polymorphic primer, with an average of 12.69 bands. The data of 376 ISSR bands were used to generate Nei’s similarity coefficients and to construct a dendrogram by means of UPGMA. The similarity coefficients data suggested great genetic diversity in genus Leymus and related diploid Triticeae species, the genetic diversity among the different species more abundant than that of the different accessions. The dendrogram and principal coordinate analysis showed explicit interspecific relationships and demonstrated close phylogenetic relationships between Leymus species and Psathyrostachys.     

DNA Polymorphisms in Lentinula edodes, the Shiitake Mushroom

DNA restriction fragment length polymorphisms (RFLPs) were examined in Lentinula edodes strains. Genomic DNA from strain 70 was cloned in plasmid vector pUC19, and 18 random clones containing low-copy DNA sequences were used to probe seven strains in Southern DNA-DNA hybridizations. Each cloned fragment revealed DNA polymorphism. An RFLP genotype was determined for each strain and the genetic relatedness was assessed. The coefficients of genetic similarity among the seven strains ranged from 0.43 to 0.90. The inheritance of RFLP markers was examined in single spore isolates. Homokaryons displayed a loss of polymorphic bands compared with the parent dikaryon. Hybrids constructed by crossing compatible homokaryons displayed the inheritance of RFLP markers from each parent homokaryon.     

Unexpected low genetic differentiation among Allopatric species of section Algarobia of Prosopis (Leguminosae)

The Chaqueña Biogeographic Province, in South America, is the main diversity centre of Prosopis. A group of sympatric species of Section Algarobia in this region constitutes a syngameon, characterised by frequent hybridization and introgression. These processes have been postulated as responsible for the low genetic differentiation observed among species within this group. In this study genetic variability and differentiation among geographically isolated species of the same section was analysed through isoenzyme electrophoresis. Variability parameters and fixation indices were estimated to determine the genetic structure of populations. Two Argentinean, P. ruscifolia (‘vinal’) and P. flexuosa (‘algarrobo amarillo’), and one North American species, P. glandulosa (honey mesquite) were studied. All of them showed, similarly to other species of the same section, high genetic variability and exhibit homozygote excess, probably due to population substructure and low rates of selfing. In contrast to our hypothesis, genetic similarity among species is not related to geographic distance. Genetic distances between P. glandulosa and South American species are similar to those observed among species of this subcontinent. The results obtained suggest that the high genetic similarity among the species of the section Algarobia studied is not due to hybridization.     

ISSR and SCoT for evaluation of hereditary differences of 29 wild plants in Al Jubail Saudi Arabian

This survey is concerned with the hereditary differences of 29 wild plants collected from fifteen different regions in Al Jubail, Saudi Arabia using two molecular marker systems, viz. inter simple sequence repeat (ISSR) and start codon targeted (SCoT) molecular markers. Ten ISSR and ten SCoT primers amplified a total of 142 and 163 bands with a 87% and 84% polymorphism, respectively. The average number of polymorphic bands for each pair of ISSR and SCoT primers combinations was 12.4 and 13.7, respectively. The highest genetic similarity for ISSR (0.97) and SCoT (0.90) were recognized between Zygophyllum qatarense-22 and Juncus rigidus-23, and between Zygophyllum qatarense-28 and Zygophyllum qatarense-29, whereas the lowest was (0.59) differentiated between Zygophyllum qatarense-6 and Salsola imbricate-18 for ISSR and between Cyperus conglomeratus-7 and Halopeplis perfoliata-14 for SCoT. This considers confirmed the value of molecular techniques such as ISSR and SCoT to assess the hereditary differences among the selected 29 weeds for hereditary preservation and plant enhancement.     

Differentiation and population subdivision in Picea mongolica based on microsatellite analyses

Picea mongolica is distributed exclusively in the eastern edge of the fixed sand dunes in Hunshandake Sandy Land, China. In this area, three groups of P. mongolica can be identified by their predominant fruit colours (purple, red and green). In this study, we used seven polymorphic microsatellites to investigate the level of genetic variation and differentiation among these groups. A significant level of genetic differentiation was detected in pairwise group comparisons. Pairwise F_ST values ranged from 0.019 to 0.028, indicating, however, a level of similarity among the groups higher than that observed in other species of the Picea genus. Cluster analysis indicated that the three groups analyzed here derived from a single gene pool. We propose that moisture differences in the sand dunes may lead to the observed differentiation in P. mongolica populations, which may be accelerated by habitat fragmentation. The observed genetic differentiation among the three groups of P. mongolica supports their classification as three different ecotypes, namely Picea mongolica f. purpurea, Picea mongolica f. rubra, and Picea mongolica f. viridis.     

Population structure and genetic diversity in bottle gourd [Lagenaria siceraria (Mol.) Standl.] germplasm from India assessed by ISSR markers

Genetic diversity analysis was undertaken in 42 geographically distant genotypes accessions of bottle gourd (Lagenaria siceraria) from India northeastern (14) and northern region (28) using inter-simple sequence repeat (ISSR) markers. A total of 209 amplified bands were obtained from 20 ISSR primers used in this study, of which 186 were polymorphic with 89.00 % band polymorphism. Various parameters namely, observed number of alleles, effective number of alleles, Nei’s gene diversity/heterozygosity, resolving power, Shannon’s information index and gene flow were estimated under experiment. Jaccard’s similarity coefficient matrix was generated for pairwise comparisons between individual ISSR profiles and UPGMA cluster analysis based on this matrix showed clustering into six groups. Jaccard’s coefficient of similarity values ranged from 0.409 to 0.847, with a mean of 0.628 revealing a moderate level of genetic diversity. The Bayesian model-based approach to infer hidden genetic population structures using the multilocus ISSR markers revealed two populations among the 42 genotypes. This is the first report on the assessment of genetic variation using ISSR markers in this medicinal vegetable plant, and this study of diversity analysis will be helpful in analyzing future hybrid breeding strategy and devising effective germplasm exploration and conservation strategy.     

AFLP analysis of genetic diversity within and between Arabidopsis thaliana ecotypes

The degree of genetic diversity within and between 21 Arabidopsis thaliana (L.) Heynh ecotypes was estimated by AFLP analysis. Within seven of the 21 ecotypes, a low but significant level of polymorphism was detected, and for five of these ecotypes two or three distinct subgroups could be distinguished. As these ecotypes represent natural populations, this intra-ecotypic diversity reflects natural genetic variation and diversification within the ecotypes. The source of this diversity remains unclear but is intriguing in view of the predominantly self-fertilizing nature of Arabidopsis. Interrelationships between the different ecotypes were estimated after AFLP fingerprinting using two enzyme combinations (EcoRI/MseI and SacI/MseI) and a number of selective primer pairs. SacI recognition sites are less evenly distributed in the genome than EcoRI sites, and occur more frequently in coding sequences. In most cases, AFLP data from only one enzyme combination are used for genetic diversity analysis. Our results show that the use of two enzyme combinations can result in significantly different classifications of the ecotypes both in cluster and ordination analysis. This difference most probably reflects differences in the genomic distribution of the AFLP fragments generated, depending on the enzymes and selective primers used. For closely related varieties, as in the case of Arabidopsis ecotypes, this can preclude reliable classification.     

Evaluation of genetic diversity in Lentinula edodes strains using RAPD, ISSR and SRAP markers

Random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR) and sequence-related amplified polymorphism (SRAP) markers were used to evaluate the genetic diversity among 23 elite Lentinula edodes strains in China. A total of 138, 77 and 144 bands were detected by 16 RAPD primers, 5 ISSR primers and 23 SRAP primer combinations, among which 58.8%, 73.5% and 56.3% was polymorphic, respectively. By UPGMA clustering, a dendrogram was constructed based on each analysis. The three dendrograms showed that 23 L. edodes strains were clustered into three or four groups. The grouping exhibited similar structure and was generally consistent with their pedigrees. Twenty-three L. edodes strains shared great similarity indicated that the low level of genetic diversity of L. edodes strains and their relationship between each other. The important source of breeding material, such as wild and exotic types, must be introduced in order to broaden genetic base and decreases genetic vulnerability of L. edodes.     

不同干扰生境下河口莲座蕨遗传多样性分析

该研究利用 ISSR 分子标记,对不同生境下的6个河口莲座蕨种群进行遗传多样性分析。结果表明：从44条ISSR引物中筛选出8条能够扩增出清晰、稳定条带的引物,对6个河口莲座蕨种群进行基因组 DNA扩增,共扩增出144条带,其中多态性条带119个,多态性条带比率为93.7％,Nei’s遗传多样性指数(H)为0.296,Shannon多样性指数(I)为0.457,遗传分化指数(Gst)为0.1520,种群遗传一致度和遗传距离分别为0.9138~0.9548和0.0584~0.0901;UPGMA聚类分析表明,种群间遗传距离与空间距离和生境类型有关。河口莲座蕨在不同干扰程度的生境中,种群具有高水平的遗传多样性,一定强度或频率的干扰生境中,种群遗传多样性较高,与海拔、坡度和坡向无相关性。