To support conservation policies for old Dutch grasslands that are still in agricultural use, morphological variation and AFLP-based (amplified fragment length polymorphism-based) genetic diversity was studied in perennial ryegrass and white clover populations and compared with the diversity in reference varieties. In addition, AFLP variation was also studied in grasslands located in nature reserves. From principal component analysis (PCA), it appeared that date of ear emergence in perennial ryegrass and characters related to plant vigour in white clover were the main morphological characters separating the reference varieties from the old Dutch grassland populations, and some of the grassland populations from each other. In both species, intrapopulation variation was lower for the reference varieties. Lower heterogeneity within the reference varieties was also found in the AFLP analysis. All common AFLP's observed in old Dutch grasslands could also be found in the reference varieties and nature reserves. Only a small number of low-frequency alleles found in old Dutch grasslands were absent from the other two groups. However, band frequencies of markers could vary considerably between populations, which may have been caused by selection. Analysis of the AFLP data by PCA distinguished the majority of reference varieties from the old Dutch grasslands, and showed genetic differentiation only between some grasslands. Comparison of old Dutch grasslands with grasslands in nature reserves indicated that basically the same range of genetic variation is covered by the two groups. Our study indicates that the Netherlands harbour a more or less continuous population for major parts of the diversity of perennial ryegrass and white clover. It was concluded that no specific conservation measures are presently needed to maintain genetic diversity of perennial ryegrass and white clover occurring in old Dutch grasslands.

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SummaryGenebank accessions stored as seed populations require periodic rejuvenation in order to maintain sufficient numbers of viable seeds. During rejuvenation the genetic composition of accessions may be altered for a variety of reasons, of which variation in pollination rates between plants is the least understood. In the present study, a paternity exclusion analysis was performed on a rejuvenated accession of perennial ryegrass. In addition, flowering data of the 49 parental plants were collected during the flowering season. The aim of the study was to determine how accurate variation in pollination rates between plants can be predicted from data on the spatial and temporal distribution of pollen release. The parental population and a total of 551 offspring from 12 progeny arrays were genotyped by means of molecular analysis. Using 25 microsatellites, paternity was identified for 81.9% of the offspring, while remaining ambiguities were resolved by AFLP analysis, except in four cases. Within the total sample 9 cases of contamination were observed. Mating within the study population was clearly non-random, as 61.9% of the identified pollen donors were located within 1 m distance from the mother plant. Observed pollination rates were very well described by an inverse quadratic function of inter-plant distance between potential mating pairs. Incorporation of the recorded flowering data in the calculation of expected pollination rates improved the goodness of fit with observed values by only 0.77%. Suggestions to reduce the variance in paternal contributions were presented. However, contamination was considered more threatening to the genetic integrity of perennial ryegrass germplasm than variation in pollination rates between plants, and indicated the need for improved measures to avoid gene flow from other germplasm.

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