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    We appreciate your feedback. OK, close. Three cuts were harvested in year 1 , four cuts in year 2 and five cuts in year 3 and year 4. The total harvest of each cut was weighted to determine the fresh weight. The botanical composition of mixed species swards was determined right before each cut by collecting four subsamples of approximately g per plot to account for local heterogeneity.

    The subsamples were separated manually into three fractions: grass, clover and weed a minor weed fraction was harvested in the first year, but was negligible in subsequent years. The portion of each fraction was averaged over the four subsamples. Leaf samples for genetic analysis were collected in after establishment of the 14 plots, and immediately before the spring cut during three subsequent years — At each sampling moment, 40 perennial ryegrass leaves were collected in each plot, rendering a total of 56 sets of 40 leaves Fig 1.

    Leaves were picked randomly, but sampling the same plant twice was avoided by maintaining a minimum distance of at least 15 cm between sampling positions. With this sampling strategy, we intended to get a representation of the genotypes present in the sward at a given moment in time. An overview of the field trial, the perennial ryegrass population samples and the pooling and replication strategy is provided. For GBS of individual plants, genotypes are called per plant, i.

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    The alternative allele frequency AAF ind in the set of 40 individual plants is the sum of alternative alleles, divided by the total number of chromosomes investigated 80 in this case. For genotyping the 56 population samples, leaf tissue was weighed and pooled in replicate. DNA was extracted from each pooled sample in total , and libraries were prepared. Thereafter, sequence read data was merged for each of the original 56 population samples, and AAF pool was measured for population genetic analysis.

    The pool-GBS procedure was validated on a set of 40 plants derived from seed composition 1 20 plants of replicate A and 20 plants of replicate B, both sampled in The 40 individual leaf samples were genotyped individually and allele frequencies were calculated Fig 1 as described below.