Assessment of genetic variation withinBrassica campestris cultivars using amplified fragment length polymorphism and random amplification of polymorphic DNA markers

Genetic relationships were evaluated among nine cultivars ofBrassica campestris by employing random amplification of polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) markers. RAPDs generated a total of 125 bands using 13 decamer primers (an average of 9.6 bands per assay) of which nearly 80% were polymorphic. The per cent polymorphism ranged from 60–100%. AFLP, on the other hand generated a total of 319 markers, an average of 64 bands per assay. Of these, 213 were polymorphic in nature (66.8%). AFLP methodology detected polymorphism more efficiently than RAPD approach due to a greater number of loci assayed per reaction. Cultivar-specific bands were identified, for some cultivars using RAPD, and for most cultivars with AFLP. Genetic similarity matrix, based on Jaccard’s index detected coefficients ranging from 0.42 to 0.73 for RAPD, and from 0.48 to 0.925 for AFLPs indicating a wide genetic base. Cluster analyses using data generated by both RAPD and AFLP markers, clearly separated the yellow seeded, self-compatible cultivars from the brown seeded, self-incompatible cultivars although AFLP markers were able to group the cultivars more accurately. The higher genetic variation detected by AFLP in comparison to RAPD was also reflected in the topography of the phenetic dendrograms obtained. These results have been discussed in light of other studies and the relative efficiency of the marker systems for germplasm evaluation.     

Characterization and genetic distance analysis of isolates of Peronosclerospora sorghi using AFLP fingerprinting

Sorghum downy mildew, caused by the obligate oomycete Peronosclerospora sorghi, has been controlled through the use of resistant cultivars and seed treatment with metalaxyl. A recent outbreak in fields planted with treated seed revealed the presence of a metalaxyl-resistant variant. Here, PCR-based methods including amplification from RAPD primers and two systems of automated AFLP analysis have been used to detect DNA-level genetic variation among 14 isolates including metalaxyl-resistant and susceptible isolates, as well as representatives of common pathotypes 1 and 3 and a new pathotype. In total, 1708 bands were detected after amplification of EcoRI/MseI fragments with 16 primer combinations. Nearly as many amplified products were observed using eight primer pairs with three-base extensions (LI-COR) as with two-base extensions (ABI-Prism genetic capillary system). Approximately 25 % of the bands were polymorphic across the 14 isolates, with the majority of differences specific to the pathotype P1 isolate. The AFLP banding patterns are consistent with metalaxyl resistance and the new pathotype having evolved from pathotype 3.     

Detection of Somaclonal Variations in Tissue Culture-Derived Date Palm Plants Using Isoenzyme Analysis and RAPD Fingerprints

Isoenzyme analysis and activities of peroxidase (PER), polyphenol oxidase (POD) and glutamate oxaloacetate (GOT) and randomly amplified polymorphic DNA (RAPD) fingerprints were used to analyze somaclonal variations in tissue culture-derived date palm plants. The frequency of somaclonal variations was found to be age dependent. Similar isoenzyme patterns for PER and GOT were detected in all analyzed plants. However, variations in activities of the three enzymes studied and in POD isoenzymes were detected. RAPD analysis showed genetic variations in approximately 4 % of the analyzed plants (70 regenerants). The genetic variations were only detected in 6- and 12-months-old cultures. It was observed also that all morphologically abnormal shoots showed genetic variations at the molecular level.     

Assessment of genetic diversity in 31 species of mangroves and their associates through RAPD and AFLP markers

Random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) markers were used to assess the genetic diversity in 31 species of mangroves and mangrove associates. Four AFLP primer combinations resulted in the amplification of 840 bands with an average of 210 bands per primer combination and 11 RAPD primers yielded 319 bands with an average of 29 bands per primer. The percentage of polymorphism detected was too high indicating the high degree of genetic variability in mangrove taxa both at inter- and intra-generic levels. In the dendrogram, species belonging to a particular family/ genus, taxa inhabiting similar habitats or having similar adaptations tended to be together. There were exceptions too; as many unrelated species of mangroves form clusters. The intrafamilial classification and inter-relationships of genera in the family Rhizophoraceae could be confirmed by molecular analysis. Both the markers RAPD and AFLP were found equally informative and useful for a better understanding of the genetic variability and genome relationships among mangroves and their associated species.     

Detection of DNA polymorphism among pea cultivars using RAPD technique

An influence of some Random Amplified Polymorphic DNA (RAPD) reaction factors on resulting banding pattern and the ability of RAPD technique to detect DNA polymorphism among six economically important pea cultivars was tested. Relatively high level of DNA polymorphism among peas was observed, using polyacrylamide/urea gels and silver staining. Altogether 13 arbitrarily designed primers produced 313 amplification products. In addition 59 polymorphisms were found. These polymorphisms can serve as potential genetic markers. RAPD data were processed using cluster analysis and plotted as dendrogram. Each tested cultivar was clearly distinguished from the others. Moreover,Pisum sativum andP. sativum subsp.arvense cultivars were separated into 2 different clusters, according to their systematic relationships.     

Deciphering genetic diversity analysis of saffron (Crocus sativus L.) using RAPD and ISSR markers

The existence of genetic diversity in Crocus sativus has globally remained a mystery till date. The study investigated PCR based DNA amplification profile of saffron using ISSR and RAPD based primers. A total of 38 amplicons were generated by ISSR primers in the range from 7 to 12 with an average of 9.50 bands per primer. 20 bands were found to be polymorphic and 18 were monomorphic with an average percentage of polymorphism as 52.48%. RAPD based amplification revealed a total 161 amplicons, 107 as polymorphic and 54 as monomorphic with an average percentage of polymorphism as 66.44%. Cumulative results of RAPD and ISSR demonstrated that Nei-Li’s similarity index ranged between 0.70 and 0.97. The results of AMOVA has revealed 9% of variance among populations and 91% of variance within populations, Φ PT was found as 0.089, which indicates existence of genetic differences though limited. In conclusion, the results indicate that saffron accessions are minimally genetically differentiated, which could be capitalized in future breeding programmes to ameliorate this precious crop.     

Application of RAPD markers for characterization of γ-ray-induced rose mutants and assessment of genetic diversity

Six parent and their 12 gamma ray-induced somatic flower colour mutants of garden rose were characterized to discriminate the mutants from their respective parents and understanding the genetic diversity using Random amplification of polymorphic DNA (RAPD) markers. Out of 20 primers screened, 14 primers yielded completely identical fragments patterns. The other 7 primers gave highly polymorphic banding patterns among the radiomutants. All the cultivars were identified by using only 7 primers. Moreover, individual mutants were also distinguished by unique RAPD marker bands. Based on the presence or absence of the 48 polymorphic bands, the genetic variations within and among the 18 cultivars were measured. Genetic distance between all 18 cultivars varied from 0.40 to 0.91, as revealed by Jaccard’s coefficient matrix. A dendrogram was constructed based on the similarity matrix using the Neighbor Joining Tree method showed three main clusters. The present RAPD analysis can be used not only for estimating genetic diversity present in gamma ray-induced mutants but also for correct identification of mutant/new varieties for their legal protection under plant variety rights.     

Simplified amplified-fragment length polymorphism method for genotyping Mycobacterium tuberculosis isolates

A simplified amplified-fragment length polymorphism (AFLP) method was developed and applied to genotype 52 Mycobacterium tuberculosis isolates. This method can be carried out using only one restriction enzyme (XhoI), one double strand adapter, and one PCR primer. The amounts of DNA and DNA polymerase, and the concentrations of primer and Mg²⁺ in the PCR step were optimized using the Basic Sequential Simplex method. AFLP analysis of the isolates generated a total of 24 differently sized bands ranging from 1537 to 121 bp, and 52 different band patterns, with a minimum of 2 and a maximum of 13 bands. The results were compared with the well-established IS6110 restriction fragment length polymorphism (IS6110-RFLP) typing method, which rendered a total of 32 differently sized bands from 1 to 12 kbp, and 52 different band patterns, with a minimum of 3 and a maximum of 15 bands. Therefore, both genotyping methods showed a discriminatory power of samples of 100%. Nevertheless, pairwise comparisons of the 1326 similarity indexes calculated for both typing methods showed a total absence of correlation between the similarity indexes of the two methods. The simplified AFLP method is expected to be more useful for genotyping M. tuberculosis isolates compared to the IS6110-RFLP method, since the former evaluates genetic variations throughout the M. tuberculosis genome. Furthermore, the relatively rapid and low-cost simplified AFLP method compares favorably to the IS6110-RFLP or conventional AFLP methods, and shows great promise for genotyping M. tuberculosis isolates, especially in developing countries or for preliminary screening.     

AFLP analysis of genetic variability in New Guinea impatiens

New Guinea impatiens ( Impatiens hawkeri) is an economically important floral crop, however, little work has been conducted to further our understanding of the genetics of this crop. In this study, we used amplified fragment length polymorphism (AFLP) technology to investigate the level of polymorphism present among 41 commercial cultivars of New Guinea impatiens, study their genetic relatedness, and assess the genetic diversity in this material. An efficient DNA extraction protocol was developed, and a total of 48 EcoRI and MseI primer combinations were used for PCR amplification. Amplification products were then subjected to polyacrylamide gel electrophoresis. The AFLP analysis showed that all 41 cultivars generated between 73 and 130 scoreable polymorphic bands per primer combination. Gower's Genetic Dissimilarity estimates for the entire set of cultivars ranged between 0.940 and 0.488. A dendogram was generated from these dissimilarity data that revealed four groupings among these 41 cultivars. The implications of these results on genotypic variation, genetic relationships, and genetic diversity in New Guinea impatiens will be discussed.     

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 phylogeny of date palm (Phoenix dactylifera L.) cultivars from Saudi Arabia by DNA fingerprinting

Genetic diversity among 13 different cultivars of date palm (Phoenix dactylifera L.) of Saudi Arabia was studied using random amplified polymorphic DNA (RAPD) markers. The screening of 140 RAPD primers allowed selection of 37 primers which revealed polymorphism, and the results were reproducible. All 13 genotypes were distinguishable by their unique banding patterns produced by 37 selected primers. Cluster analysis by the unweighted paired group method of arithmetic mean (UPGMA) showed two main clusters. Cluster A consisted of five cultivars (Shehel, Om-Kobar, Ajwa, Om-Hammam and Bareem) with 0.59–0.89 Nei and Li's coefficient in the similarity matrix. Cluster B consisted of seven cultivars (Rabeeha, Shishi, Nabtet Saif, Sugai, Sukkary Asfar, Sukkary Hamra and Nabtet Sultan) with a 0.66–0.85 Nei and Li's similarity range. Om-Hammam and Bareem were the two most closely related cultivars among the 13 cultivars with the highest value in the similarity matrix for Nei and Li's coefficient (0.89). Ajwa was closely related with Om-Hammam and Bareem with the second highest value in the similarity matrix (0.86). Sukkary Hamra and Nabtet Sultan were also closely related, with the third highest value in the similarity matrix (0.85). The cultivar Barny did not belong to any of the cluster groups. It was 34% genetically similar to the rest of the 12 cultivars. The average similarity among the 13 cultivars was more than 50%. As expected, most of the cultivars have a narrow genetic base. The results of the analysis can be used for the selection of possible parents to generate a mapping population. The variation detected among the closely related genotypes indicates the efficiency of RAPD markers over the morphological and isozyme markers for the identification and construction of genetic linkage maps.Communicated by H.F. Linskens     

RAPD markers for confirmation of somatic hybrids in the dihaploid breeding of potato (Solanum tuberosum L.)

Summary The applicability and reliability of RAPD markers were evaluated for an examination of the possible use of RAPD markers to confirm hybridity of somatic hybrids between dihaploids of potato (Solanum tuberosum L.). Most of the primers examined detected polymorphism among either tetraploids or dihaploids, and polymorphism was easily detected even among closely related clones. Most of the examples of polymorphism were confirmed as being the result of amplification from the nuclear genome by a comparison of patterns generated by PCR of clones that carried the same cytoplasm. All the bands of dihaploids were transmitted stably to the respective hybrids. In the absence of primers that generated complementary polymorphic bands for both parents, a mixture of two appropriate primers, each of which generated a band specific to one parent, permitted the simple confirmation of hybridity. Hybridity of all the fusion-derived regenerants of 32 fusion combinations was unequivocally confirmed, a result that suggests that RAPD analysis could be universally applicable to the confirmation of hybridity in the dihaploid breeding of potato.     

Genetic stability analysis of tissue culture derived date palm cv. Dedhi plants using IRAP markers

Phoenix dactylifera cv. Dedhi is an excellent soft date palm cultivar of district Khairpur, Sindh-Pakistan with large size fruit, brittle texture and very low tannins at the Khalal stage. The population of this palm cultivar is currently threatened by pest and disease issues, a limited availability of offshoots and by the prevailing monoculture of semi-dry cultivars together with import of exotic elite cultivars into the region. Hence, the current study aimed to determine genetic stability of large-scale in vitro multiplied materials after their transfer to the field. The palms were phenotyped and then genotyped using two Inter-retrotransposons Amplified Polymorphism (IRAP) markers. Thirteen IRAP primers with 24 combinations were screened, of which 14 combinations produced 301 clear and reproducible bands which were all monomorphic. The IRAP banding patterns showed no variation among the tissue culture derived plants tested. No significant variation in fruit characteristics of the tissue culture derived plants was found when compared to mother plant. Using an optimized micropropagation protocol, number of plants unable to form inflorescences was reduced from 36.7% in the year 2016 to 10% in 2018. The study confirms the current micropropagation protocol to be effective for production of true-to-type plants and the applied marker system to be efficient for validating genetic stability of in vitro produced palms.     

Assessment of genetic fidelity of micropropagated date palm (Phoenix dactylifera L.) plants by RAPD and ISSR markers assay

RAPD (Random Amplified Polymorphic DNA) and ISSR (Inter-Simple Sequence Repeats) markers assay were employed to validate the genetic stability of date palm (Phoenix dactylifera L.) plants multiplied through somatic embryogenesis with upto forty two in vitro subcultures. Out of the 160 RAPD and 21 ISSR primers screened, 30 RAPD and 12 ISSR primers produced a total of 347 (246 RAPDs + 101 ISSRs) clear, distinct and reproducible amplicons, which were monomorphic across all micropropagated plants (27) studied. Thus, a total 8592 bands (number of plants analysed x number of amplicons with all the primers) were generated which exhibited homogeneous banding patterns with both RAPD and ISSR markers. These results indicate that the micropropagation protocol developed by us for rapid in vitro multiplication is appropriate and suitable for clonal propagation of date palm and corroborated the fact that somatic embryogenesis can also be used as one of the safest modes for production of true-to-type plants.     

Effect of radiation on regeneration of Chinese narcissus and analysis of genetic variation with AFLP and RAPD markers

The aim of our study was to establish an efficient in vitro propagation protocol for Chinese narcissus (Narcissus tazetta var. chinensis) to obtain variants of this species using γ-radiation treatment and evaluate the effectiveness of this system for variant induction using amplification fragment length polymorphism (AFLP) and randomly amplified polymorphic DNA (RAPD) analysis. Various doses (5–100 Gy) of gamma rays were applied to investigate the effect of radiation on adventitious bud formation from bulb-scales and the survival rate of plantlets. It was demonstrated that the regeneration of Chinese narcissus was very sensitive to gamma radiation even at low doses. The survival and multiplication rate significantly decreased with an increase of radiation dose. The optimal irradiation dose for survival and mutation induction was approximately 10 Gy. The genetic variations among the regenerants derived from irradiated explants were evaluated by DNA fingerprinting using RAPD and AFLP markers which detected a variation frequency of 8.33% and 15.48% respectively. The high frequency of mutants detected by molecular markers indicated that treatment of in vitro cultures with γ-rays may be an effective way to improve narcissus cultivars.     

Qualitative and quantitative characterization of RAPD variation among snap bean (Phaseolus vulgaris) genotypes

Ten snap bean (Phaseolus vulgaris) genotypes were screened for polymorphism with 400 RAPD (random amplified polymorphic DNA) primers. Polymorphic RAPDs were scored and classified into three categories based on ethidium bromide staining intensity. An average of 5.19 RAPD bands were scored per primer for the 364 primers that gave scorable amplification products. An average of 2.15 polymorphic RAPDs were detected per primer. The results show that primer screening may reduce the number of RAPD reactions required for the analysis of genetic relationships among snap-bean genotypes by over 60%. Based on the analysis of the distribution of RAPD amplification, the same number of polymorphic RAPDs were amplified from different genotypes for all RAPD band intensity levels. A comparison of RAPD band amplification frequency among genotypes for the three categories of bands classified by amplification strength revealed a measurable difference in the frequencies of RAPDs classified as faint (weakly amplifying) compared to RAPD bands classified as bold (strongly amplifying) indicating a possible scoring error due to the underscoring of faint bands. Correlation analysis showed that RAPD bands amplified by the same primer are not more closely correlated then RAPD bands amplified by different primers but are more highly correlated then expected by chance. Pairwise comparisons of RAPD bands indicate that the distribution of RAPD amplification among genotypes will be a useful criterion for establishing RAPD band identity. For the average pairwise comparison of genotypes, 50% of primers tested and 15.8% of all scored RAPDs detected polymorphism. Based on RAPD data Nei's average gene diversity at a locus was 0.158 based on all scorable RAPD bands and 0.388 if only polymorphic RAPD loci were considered. RAPD-derived 1 relationships among genotypes are reported for the ten genotypes included in this study. The data presented here demonstrate that many informative, polymorphic RAPDs can be found among snap bean cultivars. These RAPDs may be useful for the unique identification of bean varieties, the organization of bean germplasm, and applications of molecular markers to bean breeding.     

Comparative study of interspecific genetic divergence and phylogenic analysis of genus <Emphasis Type="Italic">Jatropha</Emphasis> by RAPD and AFLP

Genus Jatropha with 172 species having significant economic importance belongs to the family Euphorbiaceae. There are no reports on molecular characterization and phylogenetic relationship among the species of Jatropha. Hence, the present study was undertaken to assess the extent of genetic variability that exist and also to establish phylogenetic relationship among Jatropha curcas, J. glandulifera, J. gossypifolia, J. integerrima, J. multifida, J. podagrica and J. tanjorensis using RAPD and AFLP. The percentage of loci that are polymorphic among the species studied was found to be 97.74% by RAPD and 97.25% by AFLP. The mean percentage of polymorphism (PP) was found to be 68.48 by RAPD and 71.33 by AFLP. The phylogram generated with RAPD and AFLP data showed maximum similarity. With the generated data maximum relatedness was found between J. curcas and J. integerrima this may be the reason for the success of inter hybrid crosses between these two species. Neither RAPD nor AFLP data generated in this study supports the view of J. tanjorensis, a natural interspecific hybrid between J. curcas and J. gossypifolia. The present study concludes that both RAPD and AFLP techniques are comparable in divergence studies of Jatropha species. The markers generated by RAPD and AFLP can be employed efficiently for interspecific hybrids identification, marker assisted selection and genetic resource management.     

AFLP analysis of variation in pecan somatic embryos

Before somatic embryogenesis can be applied, the genetic fidelity of cultures needs to be determined. Problematic of tissue-cultured woody species is the extensive evaluation time needed for assessments. The development of methods whereby plants could be rapidly screened for potential tissue culture-derived genetic changes would be very valuable. We evaluated the applicability of AFLP (amplified fragment length polymorphism) analysis for the assessment of genetic variability in somatic embryos of pecan [Carya illinoinensis (Wangenh.) C. Koch] and made comparisons between and within embryogenic culture lines. AFLP readily detected differences between culture lines, with 368 polymorphic loci identified. Individual culture lines generally produced somatic embryos with similar overall banding patterns. Embryos derived from the same culture line generally grouped together in a phenogram generated by UPGMA (unweighted pair-group method, arithmetic average) analysis. However, a few somatic embryos exhibited higher levels of polymorphism and failed to group with others regenerated from the same line. The relation between the detected within-line differences and their contribution to phenotypic variation is yet to be determined. Received: 9 September 1998 / Revision received: 12 November 1998 / Accepted: 8 December 1998     

A Comparison of AFLP and RAPD Markers for Genetic Diversity and Cultivar Identification in Cotton

AFLP and RAPDmarkers were employed in sixteen diploid cotton (Gossypium sp) cultivars for genetic diversity estimation and cultivar identification. Polymorphism information content (PIC) and percent polymorphism were found to be more for AFLP markers as compared to RAPD markers. Average Jaccard’s genetic similarity index was found to be almost similar using either AFLP or RAPD markers. All the cultivars could be distinguished from one another using AFLP markers and also by the combined RAPD profiles. Cultivar identification indicators like resolving power, marker index and probability of chance identity of two cultivars suggested the usefulness of AFLP markers over the RAPD markers. AFLP and RAPD analyses revealed limited genetic diversity in the studied cultivars. Cluster analysis of both RAPD and AFLP data produced two clusters, one containing cultivars of G. herbaceum and another containing cultivars of G. arboreum species. Highly positive correlation between cophenetic matrices using RAPD and AFLP markers was observed. AFLP markers were found to be more efficient for genetic diversity estimation, polymorphism detection and cultivar identification.