Studies for genetic diversity for herbicide resistance in Phalaris minor Retz.
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Date
2004
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CCSHAU
Abstract
Phalaris minor is most abundant grassy weed infesting wheat crop. Due to continuous use of herbicides the plants become resistant to these herbicides. The present study was conducted on 40 biotypes taken from different regions of Haryana state to standardize DNA extraction method and PCR amplification conditions and to analyze genetic diversity among various biotypes of P. minor. DNA was isolated using CTAB method with slight modifications. Biotype from Mundhal yielded highest amount of DNA (1404 ng/μl) and from Pirthala (lead treated) yielded lowest amount of DNA (110 ng/μl). Quality of DNA was tested
on agarose gel electrophoresis and spectrophotometer. A single discrete band of high molecular weight showed that DNA was pure, free from contaminant, intact and of high quality. The ratio of absorbance ranged from 1.74 to 1.84. Clear and reproducible bands were generated by PCR amplification conditions of 50 ng genomic DNA, 1.6 mM MgCl2, 1 unit Taq DNA polymerase, 100 μM of each dNTP’s, 1 μl of 10X reaction buffer of Taq DNA polymerase 0.2 μM of primer and 40°C of annealing temperature. Of the 21 primers screened, 15 primers showed amplification while 6 primers did not show any amplification with any of the biotype of P. minor. All 15 primers were polymorphic showing 97 per cent polymorphism.
In total, 102 bands were obtained of which 99 bands were polymorphic while 3 bands were monomorphic, generated by 15 primers. For the biotypes tested, 2 to 15 bands were obtained with an average of 4.85 per primer. Eight primers amplified 40 biotypes followed by one primer, which amplified 38 biotypes. All the biotypes were distinguishable with the combinations of polymorphic bands generated by various primers. Estimates of genetic similarity ranged from 0.489 to 0.885 indicating a high genetic variability among the biotypes. Based on the tree cluster analysis using NTSYS, the genetic variation among biotypes was high enough to group the biotypes in three clusters. First cluster comprising five biotypes (four susceptible and one resistant), second cluster has 18 biotypes (12 susceptible and six resistant) and third cluster has 17 biotypes (four susceptible and 13 resistant). The results indicate that RAPDs are efficient for grouping the resistant and susceptible biotypes i.e. most of resistant biotypes grouped into separate cluster showing that these are genetically different from susceptible biotypes. This information can be used further for identification of molecular marker for herbicide resistance gene(s) in Phalaris minor Retz.