Protocols for Chromosome Preparations: Molecular Cytogenetics and Studying Genome Organization in Coffee

Abstract

Cytological preparations from cell nuclei are required to count the number of chromosomes (including determining ploidy or aneuploidy), to investigate their morphology and organization. The results are valuable for genetic and evolutionary studies, and in breeding programs to understand species relationships, polyploidy, and potential introgression of chromosomes in hybrids between different species. Preparation of good chromosome spreads with well-separated metaphase chromosomes is the foundation of cytogenetic research including chromosomal mapping based on FISH (fluorescence in situ hybridization). FISH combined with specific locus probes correlated with molecular markers to specific chromosomes for integrating physical and linkage maps as well as studying the genetic evolution of allopolyploidization, has rarely been applied in Coffea spp. despite being a global high-value crop. Cytogenetic studies of Coffea are limited by the small size and similar morphology of the chromosomes, but FISH can help to map sequences to chromosome arms and identify individual chromosomes. This chapter presents protocols for germinating seeds and growing coffee plants involving pre-treatment and fixation of root-tips where the meristems of actively growing roots have many divisions. Mitotic metaphase chromosome preparation on microscope slides is described, as well as preparing probes of 5S and 18S rDNA to be used for FISH. The FISH experiments involve a two-step protocol with pre-treatments and setting up the hybridization on day 1 and the detection of probe sites on day 2 after overnight hybridization. A final section gives advice about visualization using a fluorescent microscope and capturing images.