Karyotypic evolution study of the genus Crotalaria L. by chromosomal banding procedures and fluorescent in situ hybridization (fish)

Author(s)

Mateus Mondin

• Document type: Doctoral Thesis
• Press: Piracicaba.
• Institution: Universidade de São Paulo (USP). Escola Superior de Agricultura Luiz de Queiroz (ESALA/BC)
• Defense date: 2003-09-26
• Advisor: Margarida Lopes Rodrigues de Aguiar Perecin

Abstract

Crotalaria is one of the largest genus in the family Leguminosae, with more than 550 species described. The genus comprises eigth botanic sections based on morphological characters, specially flower features. The genus Crotalaria includes some crops, such as C. juncea, C. paulina, C. stiplularia and C. spectabilis. The main crop is C. juncea, sunnhemp, used for fine paper and cordage production, and green manure. Most ofthe species described have 2n=16, and few species have 2n=14 or are polyploids with somatic numbers which are multiples of these numbers. Based on these observations about chromosome numbers there are two basic numbers proposed to the group, x=8 and x=7. Moreover, a restricted number of species have their karyotypes precisely described and no modem cytogenetic techniques have been applied to Crotalaria, then, features oflinear differentiation ofthe chromosomes are unknown. Five species were investigated: C.juncea (2n=16), C. stipularia (2n=32) and C. paulina (2n=32) of the section Calycinae, C. spectabilis (2n=16) of the section Crotalaria and C. incana (2n= 14) of the section Chrysocalycinae. The application of C-banding, fluorochrome banding with chromomycin A3 and 2’-4’-diamidino-2-phenylindole (DAPI) and fluorescent in situ hybridization (FISH) procedures allowed the characterization chromosomal markers, very important for karyotype evolution studies. The species belonging to sections Calycinae and Crotalaria – subsection Crotalaria have pericentromeric C-bands, CMA positive and thus GC-rich, and C-bands corresponding to secondary constriction adjacent heterochromatins, highly GC-rich by the combination CMA/distamycin (DA). ln the C. incana no C-bands could not be differentiated by the procedure used, however many thin DAPl bands were identified, as well as four GC-rich regions, being one corresponding to secondary constriction adjacent heterochromatin, another with 5S rDNA and two not related to rDNAs. FISH of probes 45S and 5S rDNAs showed signals corresponding to highly GC-rich regions. Only one signal of FlSH using 5S rDNA probes was detected in chromosome 1 of C. Juncea and C. spectabilis, and chromosome 3 of C. incana. A FlSH signal correspondente to 45S rDNA loci were identified at the secondary constriction and adjacente heterochromatic blocks in the chromosome 1 of all the species analysed. Additionally a 45S rDNA site was observed in a chromosome identified as pair 4 in C. juncea. ln the polyploid species a multiple number of 45S rDNA signal were not identified. Additionally to the signal on chromosome 1, two sites were observed in C. paulina and one in C. stipularia. ln C. incana the major 45S rDNA locus in chromosome 1 and two minor ones in chromosomes 4 and 5 were revealed. Only C. spectabilis presented one 45S rDNA locus. These results suggest that the polyploid species lose 45S rDNA loci in one of the homeologues of chromosome 4 of the ancestral species similar to C. juncea in C. paulina, and in two chromosomes corresponding to pairs 1 and 4 of such ancestral. The results suggest some trends during genome evolution of these species, such as gain and/or loss of AT and GC-rich sequences. lf one considers x=7 as a basic numberderived from x=8, chromosome rearrangements and gain of At-rich repetitive sequences could have occurred. Changes in GC-rich sequences are evident in polyploids, in which 45S rDNA loci were lost, as well as other classes of sequences not studied in the presente work, but that the smaller size of the chromosomes of the polyploids seem to evidence. (AU)