Genetic analysis of maize by using the AFLP method

Methylation AFLP® is a novel PCR-based method to detect methylation of restriction sites randomly over the genome.

In framework of the AFLP®-related technology development, a modification of the AFLP method was developed, called the methylation AFLP® method. Several features of the methylation AFLP as a novel PCR-based method to detect methylation of restriction sites randomly over the genome, are described. It is illustrated how the technique can be used 1) to estimate the extent of CpG and CpNpG methylation for maize, 2) to demonstrate that most moderately to highly repetitive DNA sequences in maize are strongly methylated and 3) to generate AFLP fragments originally bounded by a methylated restriction site, in order to study hypermethylated portions of the genome.

In support of application projects, the inheritance of AFLP markers, whether or not bounded by a methylated restriction site, relative to already known RFLP markers and isozymes, was investigated in two segregating populations of maize and outlined. To my knowledge, this is the first detailed report of mapping C-methylation and its stable transmission from parent to offspring. The efficiency of generating high-density linkage maps using the AFLP technology was evaluated in terms of multiplex ( M ), effective multiplex ( EM ) and effective mapped multiplex ( EMM ) ratio. Both genetic maps of maize could be aligned on the basis of common AFLP markers and the allele-specificity of AFLP markers across both populations could be investigated. AFLP markers generated by CNG methylation sensitive and CNG methylation insensitive enzyme combinations and AFLP markers collected from hypomethylated and hypermethylated regions were compared for their genomic distribution and their position relative to the centromere.

Aiming at further characterization of AFLP data as tool for the breeder, AFLP markers associated with different enzyme combinations and originating from different methylated genomic regions were compared for their polymorphism information content (PIC), marker index (MI) and patterns of genetic diversity among a representative sample of maize inbred lines. Furthermore, the effect of reducing marker information redundancy, the choice of enzyme combination and the bootstrap sample size on the bootstrap sample variance of marker data in the estimation of genetic similarities among inbred lines, was determined.

In contrast with the chapters mentioned so far, where the application and evaluation of the AFLP and the methylation AFLP method have been restricted to general questions encountered in many crop species, the last chapter addresses a more maize-specific issue. Identification of genetic factors contributing to hybrid performance and/or heterosis and finding a suitable method that could predict hybrid performance and/or heterosis with some accuracy before field evaluation of hybrid performance and heterosis for grain yield are of particular interest in maize breeding. A novel approach towards the prediction of heterosis and hybrid performance is presented here. This approach is based on 1) the assessment of associations between markers and hybrid performance across a number of hybrids and 2) the assumption that the joint effect of genetic factors (loci) determined this way can be obtained by addition. Since the map position of the selected markers is known, putative QTL affecting the trait of interest are identified.