BERGAMO, ITALY
Istituto Sperimentale per la Cerealicoltura
Polymorphism, distribution, and segregation of EcoRI and PstI based AFLP markers in a molecular linkage map in maize
--Ajmone Marsan, P, Castiglioni, P, Ferrarini, M, van Wiik1, R, Motto, M
1Keygene N.V., Agro Business Park 90, P.O. Box 216, 6700 AE Wageningen, The Netherlands.

Extensive genome mapping based on DNA restriction fragment length polymorphism (RFLP) markers has been accomplished in many crop species. These maps and their associated technology have been used successfully for a number of applications in genetic research and breeding, including gene tagging, evolutionary studies, marker-aided selection, and analysis of quantitative trait loci (QTLs). However, these maps and their associated QTL analyses are expensive and time-consuming technologies and may not provide detailed coverage throughout the genome.

The development of the polymerase chain reaction (PCR) has expanded the repertoire and efficiency of DNA marker systems, which include the AFLP method (Vos et al., Nucleic Acids Res. 23: 4407-4414, 1995). The advantage of AFLP assay over other DNA marker techniques includes the detection of a large number of polymorphisms from a single PCR reaction, within a very short period of time, and the requirement for small amounts of DNA, thus reducing expenses and expediting the construction of high-density linkage maps. Accordingly, with the aim of exploiting AFLP markers in a maize genome mapping program, we used the AFLP technique in order to generate and map AFLP markers using an F2 mapping population, and to investigate their inheritance and distribution associated with the use of enzyme combinations differing in sensitivity to DNA methylation.

Two-hundred-twenty-nine F3 progenies, each tracing back to an individual F2 plant, derived by crossing the maize inbred lines B73 and A7, were used. This population has been described previously to construct an RFLP linkage map (Ajmone-Marsan et al., Theor. Appl. Genet. 90:415-424, 1995). The protocol adopted for the generation of AFLP markers was essentially the same as that described by Vos et al. (1995). DNA isolated from each F3 family was digested with an EcoRI/MseI or PstI/MseI enzyme combination (ECs).

In this study we were able to detect 1568 visible bands and map 246 AFLP markers covering 2057 cM. Five gaps larger than 30 cM remained. Therefore, the efficiency of generating AFLP markers appears substantially higher relative to RFLP mapping in the same population, and the speed at which they can be generated shows a great potential for application in marker-assisted breeding. The appropriate selection of primer combinations (PC) that generate high levels of polymorphism with markers well distributed over the genome plays a crucial role. We have also observed that some primer combinations produced as many as 19 polymorphic markers distributed over as many as 9 chromosomes.

The majority of AFLP markers (89.1%) followed Mendelian segregation. They showed allelic frequencies in agreement with expectation, and were unambiguously placed on the linkage groups (72.4%). The addition of a large number of AFLP markers to the map did not disturb the original order and relative distance of the previously mapped RFLP markers. In the experiment here reported, the assay of a relatively large number of mapping progenies, the high level of informativeness of codominant scored AFLP markers and the rejection of markers with unexpected behaviour, have probably minimised the map inflation; typing errors have been credited to be in part responsible for map extension.

By adding AFLP markers, we generated a map which is 440 cM longer than the map generated with RFLP markers alone. The increase of the total map length was mainly caused by the addition of markers to telomeric regions, where RFLP markers were poorly represented. The current study indicated that PstI/MseI PCs were more efficient in detecting polymorphism than EcoRI/MseI primers. In addition, PstI AFLP markers are more randomly distributed across chromosomes and chromosome regions, while EcoRI AFLP markers clustered mainly on centromeric regions and on chromosome 1. Specific regions were observed, in which only markers produced with either PstI/MseI or EcoRI/MseI restriction enzyme combination were located (i.e. 1S, 2S, 5L, 7S and 7L). As the amplification products generated by the EcoRI/MseI AFLP technique may contain repetitive sequences, there is a higher probability of identifying EcoRI/MseI AFLP markers than PstI/MseI AFLP markers and RFLPs in highly repetitive regions near the centromeres. This may be a plausible explanation for the stronger clustering of EcoRI-based AFLP markers in the centromeres.

The more random distribution of PstI-based AFLP markers on the genetic map reported here may reflect a preferential localisation of the markers in the hypomethylated telomeric regions of the chromosomes. There is considerable evidence that hypomethylated regions of the maize genome are associated with genes (Bennetzen et al., Genome 37:565-576, 1994) and that recombination occurs primarily within genes, or perhaps unique sequences, and rarely in intergenic regions (Dooner and Martinez-Perez, The Plant Cell 9:1633-1646, 1997, and references therein).


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