M. Thoihidul Islam
Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh 02202, Bangladesh
Mohammad Rashid Arif
Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh 02202, Bangladesh
Arif Hasan Khan Robin
Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh 02202, Bangladesh
Wheat, Blast disease, Resistance domain, Molecular markers, Microsatellite markers
Variety and Species
In silico analysis
Desired list of protein-encoding domains were collected from NCBI, Ensembl Plants and Gramene databases along with their accession numbers (Ware et al., 2002; Pruitt et al., 2006; Bolser et al., 2016; For illustration of the accessions with exon-intron and flanking regions the GSDS online software was used (Guo et al., 2007). The BLAST similarity search was conducted to find out syntenic locations of disease resistance protein-encoding accession of Thatcher genotype (GenBank accession: KY064065.1) and Pita gene (GenBank accession: AY196754.1) (Bolser et al., 2016). Thatcher is a blast resistant Brazilian wheat genotype containing two R genes Rmg2 and Rmg3 (Zhan et al., 2007). After that, Pita is a rice blast resistant gene. Pita is a monogenic qualitative R gene which is also known as Rmg16 found in Japonica rice. Previously from Pita gene, Piz3 marker was developed and utilized against rice blast (Imam et al., 2014). Finally, phylogenetic analysis was performed in online software Phylogeny.fr (Dereeper et al., 2008). “HSP distribution on genome” was obtained for disease resistance protein- encoding accession of Thatcher and Pita through BLAST search.
Marker design
Eighteen NBS-LRR protein domain encoding accessions were selected from NCBI database to design primers to develop PCR-based markers. Primers were designed using online tool Primer3plus (Table 2). The Primer3plus online tool picked up forward and reverse primers with the product size between 150 and 250 bp and annealing temperature between 58oC and 61oC (Table 2, Untergasser et al., 2007). Piz3 primer referring Pita gene was collected from Gramene database (Imam et al., 2014).
Marker Validation
Collection of genotypes and extraction of DNA
A total of 18 wheat genotypes belongs to Triticum aestivum were used in this experiment (Table 3). Among those varieties BARI Gom 33 has blast resistance, Bari Gom 31-32 have moderate blast tolerance and Bari Gom 25-30 have susceptibility to blast (DGGW, 2017, Personal Communication, NCD Barma, Director, Bangladesh Wheat and Maize Research Institute). The modified Cetyl Trimethyl Ammonium Bromide (CTAB) mini-prep method was used to extract genomic DNA from the wheat leaves (Zhang et al., 2013).
PCR amplification and agarose gel electrophoresis Addbio® Taq Master Mix was used to amplify genomic DNA of 18 wheat genotypes. Each 10 µl PCR reaction included 1 µl DNA, 2 µl of each primer, 3.5 µl PCR master mix and 3.5 µl sterile distilled water. Reactions were pre-incubated for 5 min at 94°C followed by 35 cycles of amplification at 95°C for 45 s, 58-62 °C for 45 s and 72°C for 45 s in the GeneAtlas thermal cycler (Astec Co. Ltd, South Korea). Electrophoresis of PCR product was done by using 1.5% agarose
J Bangladesh Agril Univ 17(2): 161–171, 2019
Journal