Molecular characterization in different cultivars of cicer arietinum L.

Abstract

Nine cultivars of Cicer arietinum L. (chickpea) released by Bangladesh Agricultural Research Institute (BARI) viz BC1, BC-2, BC-3, BC-4, BC-5, BC-6, BC-7, BC-8 and BC-9 were investigated cytogenetically and at the molecular level using isozyme, SDS-PAGE, RAPD- and SSR-markers for authentic characterization. The nine chickpea cultivars have certain spectrum of variation for several phenotypic and agronomic traits. A prominent nucleolus was found in the interphase nuclei and prophase chromosomes after orcein staining in each cultivar. Although the nine cultivars were found to posses 2n=16 metacentric chromosomes, differed in respect of other karyotypic features such as total length of 2n chromosome complements, range of relative chromosome length and orcein staining pattern of interphase nuclei and prophase chromosomes. The number of CMA positive bands was 2 to 6 in the metaphase chromosomes of nine cultivars with percentage of GC-rich repeats ranging from 2.66 (BC-8) to 14.52 (BC-2). Each cultivar had distinct CMA- banding karyotype formula. Heteromorphicity in CMA banding revealed the occurrence of paracentric inversion in chromosome pair II of BC-2. The number of DAPI bands was ranging from 1 (BC-4) to 4 (BC-1, BC-5, BC-6, BC-7 and BC-9). DAPI bands were confined to centromeric and terminal regions of chromosomes among nine cultivars. Some DAPI bands were so unique that these chromosomes could easily be isolated from the rest and may use as marker chromosomes for the respective cultivars. Each cultivar has characteristic DAPI-banding formula. Heteromorphicity in DAPI banding pattern between homologue members indicated the occurrence of deletion. Differential staining property of satellites revealed the stain specific nature of satellited portions. The nine cultivars showed different SDS-PAGE pattern. Cultivar BC-2, BC-6, BC-8 and BC-9 could be characterized by SDS-PAGE bands on the basis of their location, size and intensity. Three isozymes system namely acid phosphatase, esterase and peroxidase were investigated of which esterase found suitable for producing distinct polymorphic band among nine cultivars. DNA from the nine chickpea cultivars was studied with 12 oligonucleotide primers and 10 microsatellite primer pairs for RAPD and SSR assay, respectively. The ten RAPD primers generated 351 distinct bands with 91.61% polymorphisms indicated highly diversed nature. In addition to polymorphism, 19 unique RAPD sequences were identified in nine chickpea cultivars. The ten SSR primer pairs generated 143 distinct bands of which 62 were considered as polymorphic (43.36% polymorphisms). Moreover, one unique SSR sequence was identified in BC-5. The combined RAPD and SSR dendrogram made BC-8 and BC-9 distinct from the rest and placed in a separate sub cluster that correlated with their phenotypic, agronomic and cytogenetical features. Therefore, each germplasm could be characterized authentically by cytogenetical and molecular analysis. This information will be helpful for characterizing each cultivar of chickpea for improved breeding program.