Shanjida Rahman
Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh
Md. Ashraful Haque
Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh
Upama Mondal
Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh
Lutful Hassan
Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh
Maize, Resistance, Wsm gene, MDMV, MCDV
Department of Genetics and Plant Breeding, Bangladesh Agricultural University (BAU) and Biotechnology Laboratory of Biotechnology Division, Bangladesh Institute of Nuclear Agriculture (BINA).Mymensingh
Variety and Species
Experiment was conducted in the net house, Department of Genetics and Plant Breeding, Bangladesh Agricultural University (BAU) and Biotechnology Laboratory of Biotechnology Division, Bangladesh Institute of Nuclear Agriculture (BINA). Nine maize genotypes (Uttaran, Duranta, BHM-5, BHM-7, BHM-9, V-92, H-981, pop corn and sweet corn) taken from Bangladesh
PCR amplification reactions for each of the SSR markers were performed in a 10 µl reaction volume containing 2 μl genomic DNA, 1 μl of 10X buffer, 0.8 μl of MgCl2, 2 μl 0f dNTPs, 0.5 μl of each forward and reverse primer, 0.2 μl of Taq polymerase and 3 μl of ddH2O. PCR amplification was performed using a touchdown profile (Ingvardsen et al., 2010) designed for the annealing temperature (Ta) of the primer pair: initial denaturation, followed by 12 cycles of 30 s at 94º C, 1 min at Ta+ 12 ºC and 1 min at 72 ºC with a reduction of the annealing temperature of 10C at each cycle, followed by 30 cycles of s at 94 ºC, 1 min at Ta and 1 min at 72 ºC, followed by a final extension.
Virus stocks were incorporated into seeds (developed from BC1-F1 progeny of a backcross between Pa405 and Oh28) through vascular puncture inoculation (VPI) method (Stewart et al., 2013a). Seeds were received as a cordial gift from Stewart. Those seeds were planted and after growing, leaves with virus symptom were collected. The virus infected leaves were used to prepare inoculation sap which was artificially inoculated into experimental materials at 3-4 leaf stage by rubbing. Carborundum powder and buffer solution were added to the sap. Plants were inoculated at total of three times at two to three days intervals. Individual plants were scored as no symptoms (0), limited symptoms (1) and severe symptoms (2) at 7, 10 and 14 days post-inoculation (dpi) with day 0 being the first inoculation date.
DNA was extracted from the young leaves from 25 days old seedlings of each genotype using the Cetyl Trimethyl Ammonium Bromide (CTAB) mini-prep (modified) method. The quality of the isolated DNA in the protocol was sufficient for PCR analysis. Three sets of primer (Table 1) were used for screening: umc1300 for detection of Wsm loci (Ingvardsen et al., 2010), MDMVgen for detection of loci responsible against MDMV and MAHP35-MCDV-s for loci against MCDV (http://www.maizegbd.org). These markers were selected based on their potentiality for population discrimination which was determined by preliminary experiment with three sets of primers
Scoring of virus symptoms had been used to calculate % infection and AUDPC (Area Under Disease Progress Curve). Percent infection is the mean for three independent replications of each line. AUDPC was calculated from means of disease ratings for each line in each replicate using trapezoid method from the time of first disease scoring. The trapezoid method is the most common way to calculate AUDPC. It is performed by using a formula devised by Campbell and Madden in 1990 or by plotting a graph of percentage of infection against time and summing the trapezoids between time intervals (http://www.ehow.com/how12033613calculateaudpc.btml). The genotype with 100% infection would posses 14 score in AUDPC. Three types of primer evolved three different types of conclusion. The first primer, umc1300 was used to identify lines carrying Wsm loci. The lines responsible against MDMV and MCDV were screened using MDMVgen primer and MAHP35-MCDV-s primer, respectively. In all above cases only the presence and absence of band will be observed. Presence of band referred the genotype as resistant and absence of band indicated the genotype as susceptible.
J Bangladesh Agril Univ 16(2): 208–214, 2018
Journal