RESUMEN
Lippia alba is a phenotypically variable tropical shrub thought to comprise a young autopolyploid complex. Chromosome numbers in L. alba include 2n = 30, 38, 45, 60, and 90. High levels of chemical and phenotypic variation associated with economic and medicinal importance were reported. However, the genetic background including chromosome composition remains under-explored. Furthermore, the occurrence of at least four ploidal levels in L. alba and the lack of data for polyploid plants in tropical areas also merit further study of L. alba. Here we assessed the chromosome composition using two new satellite repeats (CL98 and CL66) applied as FISH probes to mitotic chromosomes, and we proposed to calculate the degree of homozygosis for CL66 satDNA (named as index h) and to associate it to meiotic instability. The CL98 mapping showed few variations in both number of signals and position. However, the levels of structural homozygosity for a satellite repeat CL66 were very variable. The numbers of CL66-bearing-chromosomes were under-represented in tetraploids relative to diploids implying that CL66 arrays have been lost in tetraploid lineages as a result of increased meiotic instability. High percentage of irregularities was observed in meiotic cells, especially in polyploids. L. alba complex comprised a mixture of homomorphic and heteromorphic chromosomes. Overall, the polyploid complex presents features typical of both young and older stable polyploids. It seems that L. alba genome is still in the process of stabilization.
Asunto(s)
Lippia , Cromosomas , Diploidia , Humanos , Lippia/genética , PoliploidíaRESUMEN
Lippia alba (Verbenaceae) is one of the most studied species of the genus Lippia, mainly due to its medicinal properties. The species was described as a polyploid complex with five cytotypes. The comparison of gene expression in species with several ploidal levels needs to be conducted carefully due to possible changes in gene regulation. Quantitative reverse transcription PCR (qRT-PCR) is a widely used method for transcript abundance analyses in plants. Besides being an extremely powerful technique, relative quantification by Real-Time quantitative PCR (RT-qPCR) needs the normalization with a stable reference gene. We evaluated the stability of nine candidate reference genes in Lippia alba with different ploidal levels using NormFinder, geNorm, and RefFinder software. The product of each primer showed a single peak in the melting curve. The R2 value ranged from 0.998 to 1000 and primers efficiency ranged from 98.95% to 129%. The CIT gene came up as a stable housekeeping gene, being appropriate for studies in polyploid accessions of Lippia alba. Considering that polyploidy is widely documented in Angiosperms, the results can be used not only for further gene expression studies in L. alba but also as a possible reference gene for other polyploid complexes. Differential stability among different genes highlights the importance of the validation of reference genes used for RT-qPCR approach in polyploid studies.
