Avaliação da estabilidade genômica em acessos naturais e sintéticos de Lippia alba (MILL.) N. E. Br. (Verbenaceae)

Abstract

Lippia alba is a medicinal species with a broad phenotypic diversity, including the essential oil composition. Genetic variation is probably the main cause of this variation. The species is a polyploid complex with five chromosome numbers (2n = 30, 38, 45, 60 and 90). Due to the economic importance and the natural genetic variation, this species represents an excellent model in studies on the genomic stability. This work aimed to investigate the genomic stability of 22 natural accessions grown in vitro for 7 years and in synthetic polyploid accessions obtained from the chromosome duplication of a natural diploid access using colchicine. To analyze the genome stability of long-term cultivated plants, four replicates (three maintained in vitro and one in the field) of 22 accessions were analyzed. The size of the genome was verified by flow cytometry and eight ISSR markers checked for stability in the DNA sequence. To evaluate the effect of polyploidization, eleven synthetic polyploid plants composed of 5 tetraploids (4X) and 6 mixoploids (2X/4X) were compared with the diploid parent plant. The comparisons were based on DNA content, chromosomal count, molecular markers ISSR and SSR, and chemical profile of the essential oil. The comparison between the plants maintained in vitro and the respective plants maintained in the field showed that 13 of the 22 accessions suffered a small reduction in the size of the genome. ISSR markers detected polymorphisms in the DNA sequence ranging from 1.61 to 33.87%. Only three accessions did not present polymorphic bands. The number of polymorphic bands among the other 19 accesses ranged from 1 to 21. Analysis of the ploidy of the synthetic polyploid plants carried out on leaves and roots confirmed the stability of the tetraploid plants. The mixoploid plants presented a single G1 peak corresponding to triploid plants. The chromosome analysis showed 60 chromosomes with twelve 45S rDNA sites in the synthetic tetraploids and nine 45S rDNA sites over the 45 chromosomes observed in the synthetics triploid plants. ISSR markers showed polymorphism between the diploid parent plant and synthetic polyploid plants. The polymorphism rate varied from 1.81 to 5.45% in the tetraploid plants and from 43.63 to 56.36% in the triploid plants. Changes in the size of the microsatellite alleles were also detected. The diploid parental plant presented 10 alleles, of which 3 were shared with the triploid plants and 7 with the tetraploid plants. All triploid plants had 13 alleles, 10 of which correspond to new alleles. The number of alleles detected in tetraploid plants ranged from 8 to 11 and the number of new alleles as a consequence of polyploidization ranged from 2 to 4. The major component detected in the essential oil of the parental diploid plant and the tetraploid plants was the citral and the triploid plants was the linalool. The genetic instability detected in L. alba after seven years of in vitro culture may be due to the consequence of natural genome instability combined with long-term in vitro culture. The effects of polyploidization that result in genomic reorganization and physiological changes may explain the variation observed in synthetic polyploid plants.