Molecular Cytogenetics of Crotalaria Spp. From Bangladesh.

Abstract

Ten germplasm of Crotalaria species viz. C. pallida (Acc. No. 4250, 4803, 4805, 4806 and 4807), C. incana (Acc. No. 4790, 4801, 4804 and 4809) and C. juncea (Local) available in Bangladesh were investigated cytogenetically and at the molecular level using RAPD- and SSR- markers for their authentic characterization. The interphase nuclei and prophase chromosomes of these germplasm exhibited different types of orcein-staining patterns. Persistent nucleolus was observed in Acc. No. 4806 of C. pallida following orcein staining. Variable numbers of somatic chromosome numbers were found in Crotalaria species. In C. pallida 2n = 16 (Acc. No. 4803, 4805 and 4807) and 2n = 18 (Acc. No. 4250 and 4806) chromosomes were observed. In contrast, C. incana was found to possess 2n = 16 (Acc. No. 4801), 2n = 17 (Acc. No. 4790 and 4804) and 2n = 18 (Acc. No. 4809) chromosomes while 2n = 16 somatic chromosome number was observed in C. juncea. Somatic chromosome number found of 2n = 17 in Acc. No. 4790 and 4804 of C. incana might be originated either by intra-specific hybridization between 2n = 18 and 2n = 16 germplasm or by aneuploid origin that correlates with their phenotypic features like seedless pod formation. Thus it is suggested to avoid these two germplasm for further breeding programme. In ten germplasm of Crotalaria the variation of chromosomal length was almost negligible. Metacentric chromosomes were found in maximum germplasm indicate their symmetric nature of karyotype. In contrast, few submetacentric chromosomes were observed in several germplasm indicate relatively asymmetric nature of their karyotype. After CMA-banding, ten Crotalaria germplasm generated 31 centromeric and 34 terminal bands which indicated a tendency of accumulating GC-rich sequences at centromeric regions or chromosomal ends. The number of DAPI-bands was less than that of the CMA-band found in different germplasm of Crotalaria. Maximum terminal DAPI bands indicated a tendency of accumulating AT-rich repetitive sequences at the chromosomal ends. Few chromosomes could be used as marker of respective germplasm due to their unique DAPI-banding pattern. Fluorescent banding revealed the occurrence of genomic alteration within these germplasm. Further genomic DNA from the ten Crotalaria germplasm was studied using fourteen oligonucleotide primers and four microsatellite primer pairs for RAPD and SSR analysis, respectively. The fourteen RAPD primers generated 881 distinct bands with 95.57% polymorphisms indicating highly diverged nature of germplasm. In addition to polymorphism, 86 unique and 50 common RAPD bands were identified in ten Crotalaria germplasm. The four SSR primer pairs generated 107 distinct bands of which all were considered as polymorphic. Moreover, 19 unique SSR bands were identified among them. The dendrogram of RAPD and SSR showed that the Acc. No. 4803 of C. pallida and Acc. No. 4790 of C. incana were distinctly different from the rest and placed alone in a separate cluster that correlated with its phenotypic and cytogenetical features. Therefore, each germplasm of Crotalaria could be characterized authentically by cytogenetical and molecular analysis.