Marcadores microssatelites e ensaio elisa auxiliam na seleção de linhagens no melhoramento genético do milho

Abstract

The growing demand for increased grain production, combined with the significant reduction in productive performance triggered by pest attacks, makes corn (Zea mays) one of the main targets of transgenic technology. The transgenics currently on the market aim to protect crops from pest attack, resistance to herbicides and drought, and among other benefits that, consequently, contribute to the good performance of the genetically modified crop. Transgenic lines developed after genetic transformation play the role of donor parents during the introgression process of a new transgene in breeding programs. These donors are then crossed with recurrent parents, which constitute lines with desirable agronomic characteristics. The genotypes resulting from this crossing are crossed again with the recurrent parent, a process known as backcrossing. This conversion process can be extended until obtaining lines with satisfactory fixation of the recurrent parent's alleles and, at the same time, expressing the transgene of the donor lineage. The use of marker-assisted selection (SAM) and ELISA assays in breeding are tools that can help in the selection of lines during the breeding process. Therefore, the objective of this work was to verify the efficiency of using microsatellite molecular markers (SSR) and the ELISA assay to evaluate the rate of allele fixation and quantification of the transgenic protein, respectively, of populations, at different stages of conversion, referring to to 3 strains (L1, L2 and L3). To assess the rate of recovery by SSR, DNA extractions were conducted from each individual to be analyzed. The DNA was then subjected to amplification using SSR markers, previously selected according to the polymorphic profile between donor and recurrent parents. The obtained PCR products were evaluated in a genetic analyzer and,from the results, the inference of the rate of fixation of the alleles of the recurrent parent was made for each strain analyzed. To evaluate the expression of the transgene, the extraction of total proteins was performed. The protein extract obtained from each strain was used to quantify the target protein using competitive ELISA. The genetic similarity inferred that L1 presented a recovery rate between 98.438 and 100.00% already in the fourth generation of backcrossing and three generations of selfing (RC4S3). On the other hand, in L2 after five generations of backcrossing and two generations of selfing (RC5S2) it presented recovery of 86.111%. L3 showed recovery of 100.00% already in the third generation of backcrossing and five generations of selfing (RC3S5). The Elisa assay was efficient for detecting the protein concentrations of all analyzed individuals. Furthermore, it was found that in the L1 and L3 strains there was no variation in protein concentration between individuals at different stages of conversion, indicating that successive backcrosses did not negatively interfere with the expression of the transgene under study. However, for the samples analyzed from L2, protein detection was inefficient and protein concentration oscillation between different stages of conversion was observed.