Validação de descritores morfológicos e moleculares na identificação de cultivares de grão-de-bico para o registro de cultivares

Abstract

Chickpea (Cicer arietinum) stands as one of the world's most significant legume crops. In Brazil, chickpea cultivation has gained prominence, with the introduction of new cultivars for farmers. Seed and seedling production, processing, and commercialization in Brazil require registration in the National Cultivar Registry (RNC) administered by the Ministry of Agriculture and Livestock (MAPA). Cultivars can only be registered if they can be identified and distinguished from existing ones. In line with decree no 10.586 of December 18, 2020, and MAPA Ordinance no 502 of December 20, 2022, which allow the use of different descriptors for material characterization, this study aimed to employ molecular analysis of proteins and DNA to distinguish between chickpea cultivars for registration in the National Cultivar Registry (RNC). Ten chickpea cultivars were used, sourced from the Empresa Brasileira de Pesquisa Agropecuária (Brazilian Agricultural Research Corporation). An experiment was conducted in a greenhouse, introducing varying levels of varietal contamination alongside chickpea cultivars to validate the descriptor's potential. For morphological characterization, plants were assessed based on descriptors recommended by the International Union for the Protection of New Varieties of Plants (UPOV). Additionally, morphometric measures were employed to analyze seed characteristics using the GroundEye® system. This system facilitated quantification of geometric features, as well as color-related traits and dominances, providing a comprehensive and precise approach to chickpea seed morphological analysis. Genetic purity certification involved percentage calculation and accuracy and error determination based on evaluations by four evaluators/experts unaware of contamination percentages. Protein analysis utilized Native PAGE and SDS PAGE electrophoresis techniques. Isoenzymatic systems such as alcohol dehydrogenase, malate dehydrogenase, catalase, esterase, superoxide dismutase, storage proteins, and heat-resistant proteins were evaluated. Protein stability was also assessed under two nitrogen levels, 50 kg.ha-1 and 200 kg.ha-1. DNA analysis utilized the Simple Sequence Repeats (SSR) technique, with subsequent product separation by polyacrylamide gel electrophoresis. Chickpea cultivars can be distinguished and identified using a combination of morphological, protein, and DNA markers. Image analysis of seeds using the GroundEye® system allows precise characterization and differentiation among chickpea cultivars, proving valuable in enriching cultivar information in the RNC. Nitrogen fertilization levels did not alter protein expression. The most appropriate developmental phase for genetic purity evaluation varies depending on the cultivar. The primer TA-203 standing out as a important marker for distinguishing Brazilian chickpea cultivars, thereby contributing to their registration.