Genetic diversity and nucleotide sequence analysis of powdery mildew marker and Vf2RAD resistant gene in apple (Malus domestica) land races

Palabras clave: Apple (Malus domestica), Vf2RAD gene, Powdery mildew marker, Cloning, Sequencing


Introduction: DNA sequencing-based methods and nucleotide sequence analysis have become the most common molecular approaches currently used for molecular typing purposes and phylogenetic diversity analysis. Methods: In this study, the nucleotide sequence variations of Powdery mildew resistance gene marker (CH03c02) and the apple scab resistance gene (Vf2RAD) beside phylogenetic diversity of seven apple landraces have been investigated. The two-locus have been successfully cloned and their nucleotide sequences were determined across all studied landraces. Results: Results of sequence alignment of the Powdery mildew resistant locus (CH03c02), compared with that of the published sequence of the same locus of Discovery genotype (HiDRAS), revealed that the nucleotide variations of this locus ranged from 1 to 28 nucleotide substitutions across all seven apple landraces. Whilst, the nucleotide variations of VF2RAD ranged from 2-8 nucleotide substitutions across all the investigated landraces. The highest genetic distance (0.062) was between Amara and Barwari. Whereas, the lowest genetic distance (0.0015) was found between each of the Lubnani, Rechard, Ispartal, and the Ahmadagha. The nucleotide sequences of the two loci were concatenated and implemented to build a Neighbor-Joining tree. The seven apple landraces were successfully grouped into two main genetic clusters (C1 and C2) in the phylogenetic tree. Conclusions: It can be concluded that the cloning approach used in the current study was found to be very successful and helpful for obtaining the full nucleotide sequences of these two loci. The investigated loci were displayed nucleotide variations among the studied landraces. And, finding of these variations was allowed the distinguishing and discrimination of these landraces.


Troggio M., Gleave A., Salvi S., Chagne' D., Cestaro A., Kumar S., Crowhurst R. N. and Gardiner S. E. Apple, from genome to breeding. Tree Genet Genomics. 2012; 8 (3): 509-529.

Eccher G., Ferrero S., Populin F., Colombo L. and Botton A. Apple (Malus domestica L. Borkh) as an emerging model for fruit development. Plant Biosystems. 2014; 148(1): 157-168.

Sofla H.S., Zamani Z., Talaei A.R., Fatahi M.R., S. Nazari S.A., Farokhzad A.R., Gharghani A. and Asgarzadeh M. Introduction of New Promising Apple Genotypes: A Study of Quality Attributes of Apple in Crosses between Iranian Early Ripening and Exotic Late Ripening Apple Cultivars. International Journal of Fruit Science. 2016; 6 (59): 1-15.

Volk G.M., Chao C.T., Norelli J., Brown N.J., Fazio G., Peace C., McFerson J., Zhong G. and Bretting P. The vulnerability of US apple (Malus) genetic resources. Genet Resour Crop Evol. 2015; 62:765-794.

Muzher B. M., Younis R. A. A., El-Halabi O. and Ismail O. M. Genetic Identification of Some Syrian Local Apple (Malus sp.) Cultivars Using Molecular Markers. Res. J. Agric. and Biol. Sci. 2007; 3(6): 704-713.

Faramarzi S., Yadollahi A., and Soltani B. M. Preliminary Evaluation of Genetic Diversity among Iranian Red Fleshed Apples Using Microsatellite Markers. J. Agr. Sci. Tech. 2014; 16: 373-384.

Khadivi-Khub A., Jahangirzadeh S., Ahadi E., and Aliyoun S. Nuclear and chloroplast DNA variability and phylogeny of Iranian apples (Malus domestica). Plant Syst Evol. 2014; 300: 1803-1817.

Mratinić E. and Akšić M.F. Phenotypic Diversity of Apple (Malus sp.) Germplasm in South Serbia. Braz. Arch. Biol. Technol. 2012; 55(3): 349-358.

Poczai P. and Hyvonen J. Plastid trnF pseudogenes are present in Jaltomata, the sister genus of Solanum (Solanaceae): Molecular evolution of tandemly repeated structural mutation. Gene. 2013; 530: 143-150.

Garkava-Gustavsson L., in Swedish and finnish heirloom apple cultivars revealed with SSR markers. ScientiaHorticulturae Mujaju C., Sehic J. and Zborowska A., Backes G.M., Hietarana T. and Antonius K. Genetic diversity. 2013; 162: 43-48.

Király I., Ladányi M., Nagyistván O. and Tóth M. Assessment of diversity in a Hungarian apple gene bank using morphological markers. Org.Agr. 2015; 5:143 -151.

Gygax M., Gianfranceschi L., Liebhard R., Kellerhals M., Gessler C. and Patocchi A. Molecular markers linked to the apple scab resistance gene Vbj derived from Malus baccata jackii. Theor Appl Genet. 2004; 109: 1702-1709.

Patzak J., Paprstein F. and Henychova A. Identification of Apple Scab and Powdery Mildew Resistance Genes in Czech Apple (Malusdomestica) Genetic Resources by PCR Molecular Markers. Czech J. Genet. Plant Breed. 2011; 47 (4): 156-165.

Bus V. G. M., Rikkerink E. H. A., van de Weg W. E., Rusholme R. L., Gardiner S. E., Bassett H. C. M., Kodde L. P., Parisi L., Laurens F. N. D., Meulenbroek E. J. and Plummer K. M. The Vh2 and Vh4 scab resistance genes in two differential hosts derived from Russian apple R12740-7A map to the same linkage group of apple. Molecular Breeding. 2005;15: 103-116.

Boudichevskaia A., Flachowsky H. and Dunemann F. Identification and molecular analysis of candidate genes homologous to HcrVf genes for scab resistance in apple. Plant Breeding. 2009; 128: 84-91.

Baldi P., Patocchi A., Zini E., Toller C., Velasco R. and Komjanc M. Cloning and linkage mapping of resistance gene homologues in apple. Theor Appl Genet. 2004; 109: 231-239.

Munshi A. DNA Sequencing-Methods and applications. ( InTech Janeza Trdine 9, 51000 Rijeka, Croatia 2012.ISBN 978-953-51-0564-0.

Weigand F., Baum M. and Udupa S. DNA Molecular Marker Techniques. ICARDA, Aleppo, Syria. 1993; 51: 1-10.

Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, Buxton S, Cooper A, Markowitz S, Duran C, Thierer T, Ashton B, Mentjies P and Drummond A. Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics. 2012; 28(12): 1647-1649.

Markussen T., Kruger J., Schmidt H. and Dunemann F. Identification of PCR-based markers linked to the powdery-mildewresistance gene Pli from Malus robusta in cultivated apple. Plant Breeding. 1995; 114:530-534.

Evans K. M. and James C. m. Identification of SCAR markers linked to Pl-w mildew resistance in apple. Theor Appl Genet. 2003; 106: 1178-1183.

Huaracha E. Xu M. and Korban S. S. Narrowing down the region of the Vf locus for scab resistance in apple using AFLP-derived SCARs. Theor Appl Genet. 2004; 108: 274-279.

Cova V., Lasserre-Zuber P., Piazza S., Cestaro A. Velasco R. Durel C. E. and Malnoy M. High-resolution genetic and physical map of the Rvi1 (Vg) apple scab resistance locus. Mol Breeding. 2015; 35(16):1-13.

Pessina S., Pavan S., Catalano D., Gallotta A., G.F Visser R., Bai By., Malnoy M. and Schouten H. J. Characterization of the MLO gene family in Rosaceae and gene expression analysis in Malus domestica. BMCGenomics. 2014; 15(618): 1-12.

Wei M., Wang S., Dong H., Cai B. and Tao J. Characterization and Comparison of the CPK Gene Family in the Apple (Malus domestica) and other Rosaceae Species and Its Response to Alternaria alternate Infection. Plos one. 2016; 10(1371): 1-19.

Horres R., Zizka, G., and Weising K. Molecular Phylogenetics of Bromeliaceae: Evidence from trnL(UAA) Intron Sequences of the Chloroplast Genome. Plant Biology. 2000; 2: 306-315.

Hidayat T., and Pancoro A. Short Comunication: DNA Technology and Studies in Phylogenetic Relationship of tropical Plant: Prospect in Indonesia. Paper presented at International Conference on Mathematic and Natural Science, Facultyof Mathematic and Natural Science (ICMNS), Institute Technology of Bandung, 29-30 November 2006.

Cómo citar
Ali, S. H., Jubrael, J. M. S., & Bowsher, C. (2018). Genetic diversity and nucleotide sequence analysis of powdery mildew marker and Vf2RAD resistant gene in apple (Malus domestica) land races. Revista Innovaciencia , 6(1), 1-10.
Artículo de investigación científica y tecnológica