Analysis of CMA-DAPI bands and preparation of fluorescent karyotypes in thirty Indian cultivars of Lens culinaris

Abstract

India holds a significant rank in production and consumption of the age old protein rich crop Lentil with only one cultivated species and a large number of phenotypically similar cultivars. The need for a reliable and cost effective method of genetic characterization to unravel differences within the Lentil cultivars was felt. The present paper adopted EMA based chromosome preparation followed by staining with two contrasting fluorochromes dyes CMA and DAPI that bind directly to GC and AT rich heterochromatic segments on chromosomes. Analysis of fluorochrome banding pattern furnished a comparative account of genetic diversity within the cultivars that could not be achieved by traditional karyotyping. The marker pair of nucleolar chromosomes (4th and 3rd, majorly) occupied a pivotal position to intensify differences between cultivars in terms of banding patterns around secondary constrictions, suggestive of yet unknown variation in heterochromatin composition. Our study has strengthened genetic background and relationships of Lentil cultivars. We observed certain types of unusual fluorochrome bands that put forward the exclusivity of Indian germplasm and have questioned the mainstream heterochromatin elements of plant chromosomes captured by CMA-DAPI stains. The comprehensive fluorescent karyotypes of 30 L. culinaris cultivars prepared for the first time, serve as an archetype for the benefit of future breeding programmes in any Indian crop.

Comparative Cytogenetics and Fluorescent Chromosome Banding in Five Indian Species of Dipcadi Medik

“…Absolute and relative chromosome lengths were calculated [75]. Karyotype formulae and the respective ideogram for each of the individual species were generated using the chromosome morphometric data [76].…”

“…Giemsa-stained slides were de-stained with 70% methanol for 45 min, air dried and further used twice for two separate fluorochrome staining with 4 -6-Diamidino-2phenylindole (DAPI) and Chromomycin A 3 (CMA) following the protocol described earlier [74,76], with minor modifications. Slides were incubated in McIlvaine buffer I (0.1 M citric acid, 0.2 M Na 2 HPO 4 , pH 7.0) for 30 min and stained with 0.1 µg/mL DAPI solution for 25-30 min.…”