Makful, S. Purnomo, and Sunyoto. Analysis of Genetic Diversity of Mangosteen Basedon the Amplified Fragment Length Polymorphism (AFLP) Technique. For estimating genetic distance the AFLP and RFLP markers gave the most The dominant markers (AFLP and RAPD) had small CV values indicating a. Key Words: Aegilops, AFLP, DNA fingerprinting, durum wheat. Bu¤dayda ve Aegilops’ta çok teknik deneyim gerektirmektedir. Bununla birlikte ÇPUP.

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Garcia I ; Luciana L. Barbosa I ; Isaias O.

Comparison of RAPD, RFLP, AFLP and SSR markers for diversity studies in tropical maize inbred lines

I ; Anete P. In order to compare their relative efficiencies as markers and to find the most suitable marker for maize diversity studies we evaluated 18 inbred tropical maize lines using a number of different loci as markers. The loci used were: Bootstrap analysis were used to evaluate the afllp of loci for the markers and the coefficients of variation CV revealed a skewed distribution.

Except for the RAPD markers, all the markers correlated genetic distance with single cross performance and heterosis which showed that they could be useful in predicting single cross performance and heterosis in intrapopulation avlp for broad-based populations. Our results indicate that AFLP seemed to be the best-suited molecular assay for fingerprinting and assessing genetic relationships among tropical afp inbred lines with high accuracy.

The past limitations associated with pedigree data and morphological, physiological and cytological markers for assessing genetic diversity in cultivated and wild plant species have largely been circumvented by the development of DNA markers such as restriction fragment length polymorphisms RFLPs; Botstein et al.

However, these molecular markers have technical differences in terms of cost, speed, amount of DNA needed, technical labor, degrees of polymorphism, precision sflp genetic distance estimates and qflp statistical power of tests. Although the discrimination afpp of RFLPs in diversity studies has been well documented Smith et al. Microsatellites SSRs occur frequently in most eukaryote genomes and can be very informative, multiallelic and reproducible Vos xflp al.

The application of SSR techniques to plants depends on the availability of suitable microsatellite markers, which have been developed for species such as soybean Rongwen et al. Morgante and Olivieri stated that in soybean the amount of information given by SSR loci in relation to a comparable number of RFLP loci is given by the estimated number of alleles 4. The AFLP technique is more laborious and time consuming than RAPD methods but is also more reliable, AFLP being able to detect a large number of polymorphic bands in a single lane rather than high levels of polymorphism at each locus such as is the case for SSR methods.

Although this lower sensibility in detecting informative genotypic classes might be associated with the inability to distinguish heterozygotes from homozygotes because of binary scored AFLPs, Gerber et al.

Comparisons of different DNA markers for diversity studies in maize Hahn et al. However, in the qflp of maize, tropical and temperate populations differ from each other tkenii tropical populations usually originate from composites with higher genetic variability and, most of the time, it is difficult to allocate tropical composites to well-defined heterotic groups by phenotypic evaluation.

Due to this uniqueness, tekini markers have been very useful in genetic evaluations and assignment of tropical maize inbred lines to heterotic groups. The objectives of the study described in this paper was: Falp material and DNA isolation. Eighteen S 3 selected inbred lines from two divergent tropical maize populations eight from BR and ten from BR previously had their genetic distances surveyed using four different marker systems Lanza et al.

Detailed descriptions of these populations are given in Lanza et al. Total genomic DNA was isolated from a bulk of five-week-old leaf tissue taken from 16 plants of each line, then being isolated and purified by the method of Hoisington et al. Thirty-two primers showing reproducible polymorphism were selected and used for scoring the 18 inbred lines.

When performing RAPD analysis, each band was considered as one locus.

Briefly, a total of clone-enzyme combinations were analyzed, the maize genome being saturated 20 cM intervals with at least one RFLP probe selected by its map location on each chromosome. For the AFLP method 20 primer combinations were used and binary scored 1 or 0 with each band being considered a locus while for the SSR method 68 polymorphic primers were used with the binary data being converted into a genotypic matrix which was used to identify alleles and their respective loci.

Both dominant markers RAPD and AFLP were used to calculate the genetic distances between the 18 inbred lines using the complement of the Jaccard’s similarity coefficient Jaccard, which takes into account the presence or absence of bands.


In this method, co-occurrences are divided by the total number of evaluated loci excluding the negative co-occurrences and thus can be interpreted as the proportion of coincidences in relation to the total number of evaluated loci. Jaccard similarities were calculated using version 2. The genetic distances for the codominant markers RFLP and SSR were calculated using the modified Roger’s distance MRD; Goodman and Stuber, based on the allele frequency of each locus which considers the amount of genetic diversity and expresses the quantity of diversity present in each locus or allele, calculations being made using version 1.

Pearson’s correlation coefficient was calculated for the genetic distances, single cross performance and heterosis as previously described by Benchimol et al. The information content of each marker system was calculated for each marker and locus using the polymorphism information content PIC Lynch and Walsh, which provides an estimate of the discriminating power of a locus by taking into account not only the number of alleles that are expressed but also their relative frequencies.

Calculations were made using the following formula: Bootstrap analysis was used to verify if the number of polymorphic loci evaluated was high enough to provide accurate genetic distance estimates King et al. To determine the sampling variance of the genetic distances produced by the different molecular data sets we performed bootstrap analysis using a decreasing number of loci for codominant markers or bands for dominant markers.

For each specific number of loci or bands used the polymorphic markers were submitted to random samplings with replacement bootstrap samples and genetic distances were obtained for each bootstrap sample Tivang et al. Each band visualized on the gel was considered to be the re-sampling unit for dominant markers because for these markers each band is related to one locus. Codominant markers relate tfknii band to an allele, and therefore the boostrap was applied falp locus.

The coefficient of variation CV for all genetic distances across the bootstrap samples was estimated for each specific number of loci or bands sampled, a computer program for performing these analyses being set up using the ‘RANNUNI’ function of the SAS system Version 8. We used the median and maximum coefficient of variation values to evaluate the accuracy of the genetic distance estimates because although the mean coefficient of variation is often used in the literature tkenii is needed when dealing with molecular marker tkenii for which there is no assurance that the CVs values are distributed symmetrically.

All of the 18 maize inbred lines studied by us had previously been investigated using the four different marker systems RAPD: In the work of Lanza et al. Differences in the distribution profiles also occurred between dominant and codominant markers, with dominant markers having higher standard deviations than codominant markers. Correlations between genetic distances measured with different markers.

The RAPD markers teknik clearly the most distinct type of marker because the correlation values involving this marker were equal to or lower than 0.

As expected, the magnitude of the coefficient of variation CV values decreased atlp the number of polymorphic loci bands evaluated increased. Within each sample i. Because tenii mean is not a good indicator of central tendency for skewed data we calculated the minimum number of loci falp for an accurate representation of the genetic distances by fitting an exponential function based on the mean, median and maximum CV values of the genetic distances obtained by bootstrap sampling to the data for each marker, the results of this analysis being given in the Boxplots shown in Figure 3.

We used the median CV value to calculate the following: The results obtained based on the adjusted functions except for the mean CV shown in Figure 3 are presented in Table 3. Correlation of genetic distance with F 1 grain yield and heterosis.

Similar patterns were observed for both genetic distance and heterosis. Although similar average genetic distance values were obtained for the BR and BR arlp crosses, the BR crosses showed the widest range of genetic distances with all of the four different markers assayed; probably because of the broader genetic base of the BR population.

Brazilian breeding programs have exploited the genetic diversity of the BR population and demonstrated that high performance cultivars can be obtained from this afl Gerage et al.

Analisis Keragaman Genetik Manggis Menggunakan Teknik Amplified Fragment Length Polymorphism (AFLP)

The correlation coefficient values between genetic distance and hybrid performance for the four markers assayed were similar to the correlation values between genetic distance and heterosis, not only for the inter or intrapopulation crosses but also for all crosses combined.

The RFLP assay reflects restriction size variation spread across the genome, because the use of RFLP markers resulted in the greatest average number of alleles per locus as compared to the other marker systems tested. We found that estimates of polymorphism information content PIC based on RFLP measures had the lowest standard deviations and were the most informative.


As expected, the PIC distributions revealed that, in terms of genetic distance, dominant markers had lower levels of polymorphism as compared to codominant markers.

However, we also found that SSRs markers gave a more heterogeneous distribution for individual PIC values than RFLP markers, tdknii this might have been due afp the low number of polymorphic loci evaluated for this tekhii Barbosa et al. Although the AFLP aflpp gave the lowest mean PIC value they provided a similar degree of polymorphism information content to that provided by the RAPD markers, which agrees with the results published by Becker et al.

Comparison of the genetic distances generated by different molecular markers in diversity studies have been reported by several authors Hahn et al.

The results teknio in our study showed high agreement between RFLP and AFLP genetic distance estimates, such estimates having also been highly correlated in other studies Russell et al.

Indeed, we found that the RFLP and AFLP markers produced sufficient numbers of polymorphic bands to produce reliable genetic distance estimates with high correlations between these two marker systems, the similarity between the results being explainable by the fact that they are similar techniques based on restriction site changes.

Even though the CV values were not low enough to indicate a high level of precision the SSR markers geknii high, and the RAPD markers moderate, correlations between the genetic distance estimates and hybrid performance and heterosis for the BR intrapopulational crosses.

Arlp results points zflp the need to adopt different strategies for selecting markers and choosing an upper number of SSR and RAPD markers. Because the box-plots for each of our groups of samples were skewed we used the mean, median and maximum CV values to determine the adequate number of polymorphic loci needed for acceptable precision.

The box-plots Figure 3 show what happens when the genetic distance CV values, which feknii different for dominant and codominant markers, are high. The choice of the appropriate number of polymorphic loci required for a reliable estimation of genetic distance is influenced by the criteria used, and it appears that the maximum and median CV values are the best choice for evaluating the precision of the genetic distance estimates based on molecular marker data sets.

From the analysis of our data it appears that the maximum CV value appears to be, in most cases, the best guarantee for producing reliable estimates of genetic distance. For dominant markers, where the distribution is skewed towards lower genetic distance values, the use of mean or median CV values may lead to errors because some of the genetic distance values will not tekjii within the required level of precision.

For codominant markers, however, the distribution of values within each sample is skewed towards the higher values and it appears that mean or median CV values should be appropriate. Our results indicate that, apart from the RAPD markers, the other DNA marker systems provided consistent information for diversity studies on tropical maize populations and produced genetic distance estimates which were in good agreement. The RFLP system appears to be the most robust marker assay in terms of the amount of polymorphism surveyed, although, in practice, it is still a very laborious technique.

The SSR markers were promising in terms of the polymorphism and information content revealed, but may involve some additional initial costs associated with primer development. The results also suggest that the number of loci evaluated should be increased. Our results suggest that AFLP markers are the best choice for the evaluation of diversity and assessing the genetic relationships between tropical maize inbred lines with high accuracy.

The AFLP system presents good levels of precision in its genetic estimates and single crosses prediction. AFLP also correlates highly with results obtained using the RFLP system and is a fast and reliable system capable of supporting a multiplex approach not requiring previous knowledge of DNA sequencing.

Theor Appl Genet Mol Gen Genet Heterotic group assignment and hybrid performance determined by RFLP marker. Plant Breeding 6: Am J Hum Genet Informe de Pesquisa VI Isozyme variation among races of maize in Bolivia. Bull Soc Vaud Nat Theor Appl Genet 94, Comparison of pedigree and RFLP data.

The Plant J 3: Rev Bras Genet 4: Ag Biotech News Inf 6: J Genet Breed SAS language guide for personal computers. Theor Applied Genet Comparisons with data from RFLPs and pedigree. The principles and practice of statistics in biological research, 3 rd edn. Freeman and Company, New York, pp