ГЕНЕТИКА РАСТЕНИЙ
УДК 575.17:634.11
EVALUATION OF GENETIC VARIABILITY IN CHOOSEN APPLE (Malus x domestica Borkh.) CULTIVARS BY ISSR-PCR ANALYSIS
© 2010 M. Smolik, O. Krzysztoszek
Department of Horticultural Plant Breeding, West Pomeranian University of Technology, Szczecin 71-424, Poland; e-mail: msmolik@zut.edu.pl
Received April 07, 2009; in final form, January 12, 2010
The aim of the study was to determine the genetic variability in eight apple cultivars: Delikates, Cortland, James Grieve, Lired, Jonathan, Golden Delicious, Jonagold and Idared from the collection of Fruit Growing Research Station in Rajkowo of the West Pomeranian University of Technology, Szczecin. The cultivar Delikates was obtained from the crossing of two cultivars: Cortland and James Grieve, whereas cultivar Lired is a James Grieve's sport. The second one cultivar — Jonagold was obtained from the crossing of Jonathan and Golden Delicious. The cultivar Idared is a hybrid obtained from the crossing of Jonathan and Wagener. Out of 40 primers, 17 were chosen for the final study. Those amplified a total of 183 loci (872 amplicons) out of which 34 (18.5%) were monomorphic, 128 (69.5%) were polymorphic and 22 (12%) cultivar-specific. Specific ISSR products were detected for each apple cultivar. A dendrogram was constructed using the UPGMA method which revealed two distinct clusters: I — Delikates, Cortland, James Grieve and Lired, II — Jonathan, Golden Delicious, Jonagold and Idared. Genetic similarity between Delikates, Cortland and James Grieve was 68.6, 70.8%, respectively and between cultivar Jonagold, Jonathan and Golden Delicous was 79.8, 85.2%, respectively.
Apple trees are among the most important commercial fruit trees grown in moderate climate zones. A large number of cultivars available on the market are the result of free pollination of breeding trees, controlled hybridization and somatic mutation. Numerous apple tree cultivars are allogamous forms, often of an unknown origin. In practice, they are obtained by vegetative propagation. Their monoclonic character and the fact that a large number of apple tree cultivars were formed by mutation indicate that inter- and in-tra-cultivar genetic variability is reduced to a minimum within their range [1].
Thus, a thorough genetic characterization of the afore-mentioned apple tree forms is important for at least two reasons; they are very useful for breeding programs, as well as for copyright protection of the newly obtained cultivars [2].
Traditional methods of characterising and estimating the genetic diversity of plants are based on the analysis of morphological, phenological and agronomical features. It is well known, however, that these parameters are significantly modified by environmental conditions. For many years, a precise description of this variability has been carried out using modern molecular biology to elucidate the differences underling this genetic diversity [3].
The ISSR-PCR method is an analytical technique, by which one can precisely characterise genetic variability at the level of DNA. It is based on the anal-
ysis of variability within the range of highly polymorphic microsatellite sequences [4].
According to [5], microsatellite sequences contain a higher number of random mutations then other regions of the genome. The most likely reason for these mutations is due to the inherent error rate of DNA polymerases. This may be, among other things, the result of incorrect copying of the chromosomes by DNA polymerase (slippage of DNA polymerase), which could take place in the interphase prior to each division of the cell [6]. According to the literature, the height mutation rate of microsatellite sequences eventually results in a series of insertions or deletions resulting in changes within the repeated block of sequences or in the number of its repeats.
The studies showed that the microsatellite sequences occur in introns, exons, centromeric and te-lomeric regions of chromosomes [7]. Thus, we believe that our choice of research methods for the molecular characterization of the two groups of apple trees are justifiable. These methods allow for the characterization of a full spectrum of transcribable DNA variability (polymorphism of ISSR amplicons) at the level of genes or the sequences in their vicinity. These microsatellite markers follow Mendelian inheritance patterns of independent assortment [8].
The aim of this study was to determine genetic variability within the microsatallite sequences in a sport Lired selected for the study as well as the components of parental cultivars Delikates and Jonagold. More-
over, the purpose of this work was to attempt to verify a co-dominant character of ISSR markers and then to use them in apple breeding for a MAS selection.
MATERIALS AND METHODS
Eight apple cultivars: Delikates, Cortland, James Grieve, Lired, Jonathan, Golden Delicious, Jonagold and Idared from the collection of the Fruit Growing Research Station in Rajkowo of the West Pomeranian University of Technology, Szczecin were used in this study. The cultivar Delikates was obtained from the crossing of two cultivars: Cortland and James Grieve, whereas the cultivar Lired is a James Grieve's sport. The second cultivar — Jonagold was obtained from the crossing of two cultivars: Jonathan and Golden Delicious. The cultivar Idared is a hybrid obtained from the crossing of Jonathan and Wagener. Replicate DNA extraction from young leaves of four-year old apple trees was used to assess the reproducibility of band patterns (Genomic Mini AX Plant — A&A Biotechnology). Contaminating RNA was removed by digestion with RNase A (10 mg • ml-1). 40 ISSR primers were tested during initial screening. The primers were 17- to 18-mers based on various di-, tri-, tetra- and pentanucle-otide repeats. They were anchored at the 3' end. The sixteen primers that gave the most informative patterns were used to characterise diversity of the apple culti-vars (Table 1). ISSR amplification: PCR mixtures (25 |l) contained: 1.5 mM MgCl2, 100 mM KCl, 20 mM Tris-HCl, pH 8.3, 0.1% Triton X-100, 0.2 |M primer (UBC — University of British Columbia primer sets), 0.2 mM of each dNTP, 1.0 units of Taq DNA polymerase (Fermentas MBI) and 50 ng template genomic DNA. DNA was amplified using a Mastecycler (Eppendorf) thermal cycler and using the following programme: initial denaturation at 94°C for 7 min, 40 cycles of 30 s at 94°C, 50 s at annealing temperature, 2 min at 72°C, and 7 min at 72°C for a final extension. Amplified products were mixed with 6x Orange Loading Dye Solution and were analysed by electrophoresis on a 2% agarose (Basica LE — Prona). O'RangeRuler 200 bp DNA Ladder (Fermentas MBI) was used as a size marker (3000—200 bp). For ISSR data analysis, the relative mobility position of all bands present in each analyzed apple cultivar was calculated and transformed in a data matrix in which the character "1" means the presence of a specific band and "0" represents its absence and similarity index matrices were generated based on number shared fragments. Similarity index S = 2Nab/(Na + Nb), where Na and Nb represent a total number of bands present in lanes a and b, respectably, and Nab is the number of bands shared by both lanes [9].
Rooted phenograms was constructed by cluster analysis using the UPGMA (unweighted pair group with arithmetic mean) option of the TREECON software package. UPGMA is a straightforward method of tree construction. Its original purpose was to construct taxonomic phenograms, which are trees that reflect
the phenotypic similarities between operational taxonomic units (OTUs). The method uses a sequential clustering algorithm, in which local homology between OTUs is identified in order of similarity, and the tree is built in a stepwise manner. The two OTUs that are most similar to each other are first determined and then these are treated as a new single composite OTU. Subsequently from among the new group of OTUs (composite and simple), the pair with the highest similarity is identified and clustered. This continues until only two OTUs are left. The algorithm assumes that the two most closely related OTUs are more similar to each other than they are to any other. If this is not the case, spurious results may occur. Slightly different clustering may also be seen when the data is presented to the algorithm in a different order [10]. Robustness of the tree topology was assessed by 2.000 bootstrap resamplings. Bootstrapping is a technique for estimating the reliability of an internal branch of a phylogenetic tree by resampling the original data set. With DNA sequences the bases at each position are randomly sampled then returned to the pool so that they may be res-ampled again. The bootstrap value for a branch is the percentage of such resamplings [typically 100 to 1000 (2000)] that recover the branch [10].
RESULTS
In the present study, molecular characterization of eight apple tree cultivars was carried out based on the ISSR-PCR technique using a set of 40 microsatellite primers. Out of the mentioned primers, 17 (802, 809, 810, 816, 817, 818, 820, 821, 827, 840, 844, 845, 849, 859, 860, 873 and 880) generated clear products visualized on agarose gels. In the ISSR reactions, 184 loci (872 amplicons) were amplified for eight apple tree cultivars ranging in length from ~220 bp (primer 845) to ~2530 bp (primer 827). Among them, 34 (18.5%) were monomorphic, 128 (69.5%) were characterized as polymorphic, and 22 (12%) were cultivar-specific (Table 1).
The cultivar-specific products were amplified with 11 primers. Most of them were generated with primers: 880 (5), 820 (4) and 845 (3). With regard to the character of the amplicons in this experiment, it was found that the loci defined as polymorphic for the two groups compared in each of the four apple tree cultivars could have cultivar-specific characteristics in the analyses of the individual groups. Thus, as in the case of monomo
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