Development of intron targeted amplified polymorphic markers of metal homeostasis genes for monitoring their introgression from Aegilops species to wheat
The identification of transfers of useful alien genes for metal homeostasis from non-progenitor Aegilops species using the widely available anchored wheat SSR markers is difficult due to their lower polymorphism with the distant related wild species and the lack of locus specificity further restricts their application. The present study deals with the development of intron targeted amplified polymorphic (ITAP) markers for the metal homeostasis genes present on chromosomes of groups 2 and 7 of Triticeae. The mRNA sequences of 27 metal homeostasis genes were retrieved from different plant species using NCBI database and their BLASTn was performed against the wheat draft genome sequences in Ensemblplants to get exonic and intronic sequences of the corresponding metal homeostasis genes in wheat. The ITAP primers were developed in such a way that they would anneal to the conserved flanking exonic regions of the genes and amplify across highly variable introns within the PCR limits. The primers led to the amplification of variable intronic sequences of genes with polymorphism between non-progenitor Aegilops species and the recipient wheat cultivars. Further, the polymorphic ITAP markers were used to characterize the transfers of metal homeostasis genes from the non-progenitor Aegilops species to the BC2F5 wheat-Aegilops derivatives, developed through induced homoeologous pairing. The derivatives with significant percent increase in grain Fe and Zn content over the elite cultivar PBW343 LrP showed the introgression of some of the useful Aegilops alleles of the metal homeostasis genes. The use of different metal homeostasis genes using this approach is the first report of the direct contribution of the genes for increasing the grain micronutrient content for developing biofortified wheat lines with reduced linkage drag.
KeywordsAegilops ITAP Metal homeostasis genes Induced homoeologous pairing Biofortification iron and zinc Mono 5B
The authors acknowledge the Department of Biotechnology, Government of India for Grant (BT/AGR/Wheat Bioforti/PH-II/2010) through a network project “Biofortification of wheat for micronutrients through conventional and molecular approaches—phase-II”. The authors also acknowledge the Akal College of Agriculture for providing infrastructural facilities to carry out this work.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
- Okamoto M (1957) Asynaptic effect of chromosome V. Wheat Inf Serv 5:6Google Scholar
- Rozen S, Skaletsky H (2000) Primer3 on the WWW for general users and for biologist programmers. In: Krawetz S, Misener S (eds) Bioinformatics methods and protocols: methods in molecular biology. Humana Press, Totowa, pp 365–386Google Scholar
- Sears E (1956) The transfer of leaf-rust resistance from Aegilops umbellulata to wheat. Brookhaven Symp Biol 9:1–21Google Scholar
- Sheikh I, Sharma P, Verma SK, Kumar S, Malik S, Mathpal P, Kumar U, Singh D, Kumar S, Chugh V, Dhaliwal HS (2016) Characterization of interspecific hybrids of Triticum aestivum x Aegilops sp. without 5B chromosome for induced homoeologous pairing. J Plant Biochem Biotechnol 25(1):117–120CrossRefGoogle Scholar
- Verma SK, Kumar S, Sheikh I, Sharma P, Mathpal P, Malik S, Kundu P, Awasthi A, Kumar S, Prasad R, Dhaliwal HS (2016b) Induced homoeologous pairing for transfer of useful variability for high grain Fe and Zn from Aegilops kotschyi into wheat. Plant Mol Biol Report 34(6):1083–1094CrossRefGoogle Scholar
- Xiong F, Liu J, Zhong R, Jiang J, Han Z, He L, Li Z, Tang X, Tang R (2013) Intron targeted amplified polymorphism (ITAP), a new sequence related amplified polymorphism-based technique for generating molecular markers in higher plant species. Plant Omics J 6(2):128Google Scholar