Cross genera amplification of ginger EST-SSRs in large cardamom using genomic DNA isolated from standardized simplified protocol

Abstract

Large cardamom (<em>Amomum subulatum </em>Roxb.) is one of the most important cash crops of Sikkim. The issues crippling its production have been largely addressed through improved agronomic practices but efforts for genetic improvement have not been made. Being an orphan crop with regard to its genomic resources, the present study was carried out to standardize DNA isolation protocol for large cardamom using minimal resources and cross amplification of ginger expressed sequence tag (EST) based simple sequence repeat (SSR) markers in large cardamom. The DNA isolation protocol was standardized through various modifications in the general cetyltrimethyl ammonium bromide (CTAB) procedure. The DNA isolated through standardized protocol was of high quality confirmed through both electrophoretic (clear and intact bands) and spectrophotometric studies (A₂₆₀/A₂₈₀ratio 1.68 to 1.97). The isolated DNA of all the six large cardamom samples was employed for PCR studies with 73 EST-SSR primers of ginger, out of which 18 showed cross amplification. Out of 18 primers, only 5 exhibited polymorphism showing maximum of 2 alleles per locus. In total, the PIC ranged from 0 to 0.63. A total of 23 alleles were amplified with average of 1.3 alleles per marker. A null allele marker was also observed. The results indicated low cross amplification rate (24.6%). DNA isolation protocol standardized in the study can be used across labs for extraction of quality DNA with minimal resources and primers showed cross-amplification may be further used for various molecular studies in large cardamoms.