Molecular, physiological and morphological analysis of waterlogging tolerance in clonal genotypes of Theobroma cacao L.

Abstract

In soil, anoxia conditions generated by waterlogging induce changes in genetic, morphological and physiological processes, altering the growth and development of plants. Mass propagation of cacao (Theobroma cacao L.) plantlets (clones) is affected by waterlogging caused by heavy rains and irrigation methods used to induce rooting. An experiment was undertaken to assess the effects of a 45-day flooding (anoxia) on physiological and morphological traits of 35 elite cacao genotypes, aiming at potentially identifying those with greater tolerance to flooding of the growth substrate. Eighteen fluorochrome-labeled microsatellite (SSR) primer pairs were used to assess genetic variability among clones, with 248 alleles being amplified and used to calculate similarity coefficients. The resulting dendrogram indicated the presence of four major groups, in which two represented 60% and 31% of the genotypes tested. A general trend toward high levels of heterozygosity was also found for physiological and morphological traits. The survival index (IS) for flood tolerance observed varied from 30 to 96%. Clones TSA-654, TSA-656, TSA-792, CA-1.4, CEPEC-2009 and PH-17 showed an IS value above 94%, whereas CEPEC-2010, CEPEC-2002, CA-7.1 and VB-903 clones were those mostly affected by waterlogging, with IS value below 56%. All genotypes displayed lenticel and adventitious root formation in response to waterlogging, although with different intensities. To determine whether patterns of physiological response could be associated with tolerance to anoxia, a similarity-grouping analysis was performed using the ratio between waterlogged and control values obtained for a series of physiological variables assessed. No specific pattern of physiological and morphological responses to waterlogging was strictly associated with survival of plantlets. However, results revealed by the dendrogram suggest that absence of leaf chlorosis may be a proper trait to indicate cacao clones with higher survival rates under flooding conditions. Consequences of these findings are discussed in the context of developing improved strategies for mass production of clones from elite cacao genotypes.

Genetic molecular diversity, production and resistance to witches' broom in cacao clones

“…The primers were pre-developed (Lanaud et al 1999, Risterucci et al 2000 and used to amplify the samples individually and according to a previously described protocol (Santos et al 2007). The amplification products were separated by gel electrophoresis in denaturing polyacrylamide at 5% in an ABI 377 automatic sequencer (Bertolde et al 2009). GeneScan and Genotyper programs were used to characterize the microsatellites, which were amplified from primers marked with fluorochromes and analyzed by means of the fluorescence detection system in DNA sequencer ABI 377.…”

Genetic mapping of Theobroma cacao (Malvaceae) seedlings of the Parinari series, carriers of the lethal gene Luteus-Pa

“…The 35 genomic SSRs tested among the genitors generated 83 alleles, an average of 2.44 alleles per locus, more than observed by Leal et al (2008) and Bertolde et al (2010). They used microsatellite markers.…”

“…They used microsatellite markers. According to Bertolde et al (2010), high-resolution gels, such as the 5% polyacrylamide gel that we used, make it possible to correctly identify the alleles at each locus, because it allows separation of fragments differing by only 1 bp. Also, these markers facilitate this type of analysis, without environmental influence (Lerceteau et al, 1997).…”

“…Bertolde et al (2010) reported that T. cacao genotypes have a mean heterozygosity of 40 to 67%. The two genotypes that we evaluated showed 54% heterozygous loci (Table 2).…”

“…The reagent concentrations used for the amplification of genomic SSR were those used by Bertolde et al (2010); the concentrations used for SSRs with ESTs were the same as used by Lima (2007); the concentrations for the RAPDs were those used by Leal et al (2008), with some modifications. The amplifications with RAPD primers were carried out with the thermal cycler PTC 200 model (MJ Research).…”